Multiple proteases deficient filamentous fungal cells and methods of use thereof

ABSTRACT

The present disclosure relates to compositions and methods useful for the production of heterologous proteins in filamentous fungal cells.

This application was filed under 35 U.S.C. § 371 and claims priority toInternational Application No. PCT/EP2014/064820 filed Jul. 10, 2014, andclaims priority to EP 13176001.9 filed Jul. 10, 2013, which are herebyincorporated by reference into this disclosure in their entirety.

FIELD OF THE INVENTION

The present disclosure relates to compositions and methods useful forthe production of heterologous proteins in filamentous fungal cells.

BACKGROUND

Posttranslational modification of eukaryotic proteins, particularlytherapeutic proteins such as immunoglobulins, is often necessary forproper protein folding and function. Because standard prokaryoticexpression systems lack the proper machinery necessary for suchmodifications, alternative expression systems have to be used inproduction of these therapeutic proteins. Even where eukaryotic proteinsdo not have posttranslational modifications, prokaryotic expressionsystems often lack necessary chaperone proteins required for properfolding. Yeast and fungi are attractive options for expressing proteinsas they can be easily grown at a large scale in simple media, whichallows low production costs, and yeast and fungi have posttranslationalmachinery and chaperones that perform similar functions as found inmammalian cells. Moreover, tools are available to manipulate therelatively simple genetic makeup of yeast and fungal cells as well asmore complex eukaryotic cells such as mammalian or insect cells (DePourcq et al., Appl Microbiol Biotechnol, 87(5):1617-31). Despite theseadvantages, many therapeutic proteins are still being produced inmammalian cells, which produce therapeutic proteins withposttranslational modifications most resembling the native humanproteins, whereas the posttranslational modifications naturally producedby yeast and fungi often differ from that found in mammalian cells.

To address this deficiency, new strains of yeast and fungi are beingdeveloped that produce posttranslational modifications that more closelyresemble those found in native human proteins. Thus, there has beenrenewed interest in using yeast and fungal cells to express more complexproteins. However, due to the industry's focus on mammalian cell culturetechnology for such a long time, the fungal cell expression systems suchas Trichoderma are not as well established as mammalian cell culture andtherefore suffer from drawbacks when expressing mammalian proteins.

Thus, a need remains in the art for improved filamentous fungal cells,such as Trichoderma fungus cells, that can stably produce heterologousproteins, such as immunoglobulins, preferably at high levels ofexpression.

SUMMARY

Described herein are compositions including filamentous fungal cells,such as Trichoderma fungal cells having reduced or no detectableactivity of at least three proteases, and having a recombinantpolynucleotide encoding a heterologous polypeptide that is produced atincreased levels. Further described herein are methods of improvingheterologous polypeptide stability and methods of making heterologouspolypeptides in which the proteases do have the reduced activity.Further described herein are compositions including filamentous fungalcells, such as Trichoderma fungal cells, having reduced or no detectableactivity in one or more of the following proteases: pep9, amp1, amp2,and sep1.

Thus one aspect includes filamentous fungal cells comprising at leastone endogenous protease having reduced or no protease activity, and arecombinant polynucleotide encoding heterologous polypeptide, whereinthe cell has reduced or no protease activity in one or more of thefollowing proteases: pep9, amp1, amp2, and sep2. In one specificembodiment, the production level of the polypeptide is at least two-foldhigher than the production level of the same polypeptide as produced ina corresponding filamentous fungal cell in which the proteases do nothave the reduced activity.

In another aspect, that may be combined with the precedent embodiment,it includes filamentous fungal cells having reduced or no detectableactivity of at least three proteases, where the cell further contains arecombinant polynucleotide encoding a heterologous polypeptide producedat a level of at least 2-fold higher than the production level of thepolypeptide in a corresponding parental filamentous fungal cell in whichthe proteases do not have the reduced activity. In certain embodiments,when the cell is an Aspergillus cell, the total protease activity isreduced to 50% or less of the total protease activity of thecorresponding parental Aspergillus cell in which the protease do nothave reduced activity.

In one embodiment that may be combined with the preceding embodiments,the total protease activity of the filamentous fungal cell is reduced to49% or less, 40% or less, 31% or less, 6% or less, of the total proteaseactivity of the corresponding parental filamentous fungal cell in whichthe proteases do not have the reduced activity.

In certain embodiments, the expression level of at least three proteasesis reduced or eliminated. In certain embodiments, genes encoding thethree proteases each comprise a mutation that reduces or eliminates thecorresponding protease activity. In certain embodiments that may becombined with the preceding embodiments, the three protease encodinggenes are pep1, tsp1, and slp1. In other embodiments, the three proteaseencoding genes are gap1, slp1, and pep1.

In certain embodiments, the fungal cells have reduced or no detectableactivity of four endogenous proteases; genes encoding the four proteaseseach comprise a mutation that reduces or eliminates the correspondingprotease activity. In certain embodiments that may be combined with thepreceding embodiments, the four protease encoding genes are pep1, tsp1,slp1, and gap1.

In certain embodiments that may be combined with the precedingembodiments, the three or four protease encoding genes are selected frompep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1, slp1, slp2,slp3, slp7, gap1, and gap2. In certain embodiments that may be combinedwith the preceding embodiments, the three or four protease encodinggenes are selected from pep1, pep3, pep4, tsp1, slp1, slp2, gap1, andgap2. In certain embodiments, the three or four protease encoding genesare selected from pep1, pep2, pep3, pep4, pep5, gap1, gap2, slp1, slp2,and tsp1.

In other embodiments, the fungal cells have reduced or no detectableactivity of five endogenous proteases; genes encoding the five proteaseseach comprise a mutation that reduces or eliminates the correspondingprotease activity. In certain embodiments that may be combined with thepreceding embodiments, the five protease encoding genes are pep1, tsp1,slp1, gap1, and pep4. In other embodiments, the five protease encodinggenes are pep1, tsp1, slp1, gap1, and gap2.

In certain embodiments, the fungal cells have reduced or no detectableactivity of six endogenous proteases; genes encoding the six proteaseseach comprise a mutation that reduces or eliminates the correspondingprotease activity. In certain embodiments, the cell has six proteaseencoding genes, each of which comprise a mutation that reduces oreliminates the corresponding protease activity, and the six proteaseencoding genes are pep1, tsp1, slp1, gap1, gap2, and pep4.

In certain embodiments that may be combined with the precedingembodiments, the fungal cells have three to six proteases having reducedor no detectable activity in each of the three to six proteases selectedfrom pep1, pep2, pep3, pep4, pep5, tsp1, slp1, slp2, slp3, gap1, andgap2.

In certain embodiments that may be combined with the precedingembodiments, the cell has seven protease encoding genes, each of whichcomprise a mutation that reduces or eliminates the correspondingprotease activity, and the seven protease encoding genes are pep1, tsp1,slp1, gap1, gap2, pep4, and pep3.

In certain embodiments that may be combined with the precedingembodiments, the cell has eight protease encoding genes, each of whichcomprise a mutation that reduces or eliminates the correspondingprotease activity, and the eight protease encoding genes are pep1, tsp1,slp1, gap1, gap2, pep4, pep3, and pep5.

In certain embodiments that may be combined with the precedingembodiments, the fungal cell has an additional protease having reducedactivity, the gene encoding the additional protease comprises a mutationthat reduces or eliminates the corresponding protease activity, and theadditional protease is selected from pep7, pep8, pep11, pep12, tpp1,gap2, slp3, slp5, slp6, slp7, and slp8.

In certain embodiments that may be combined with the precedingembodiments, the cell has eight protease encoding genes, each of whichcomprise a mutation that reduces or eliminates the correspondingprotease activity, and the eight protease encoding genes are either

-   -   a) pep1, slp1, gap1, gap2, pep4, pep3, pep5, amp1,    -   b) pep1, slp1, gap1, gap2, pep4, pep3, pep5, amp2,    -   c) pep1, slp1, gap1, gap2, pep4, pep3, pep5, sep1,    -   d) pep1, slp1, gap1, gap2, pep4, pep3, pep5, pep9, or    -   e) pep1, slp1, gap1, gap2, pep4, pep3, pep5, pep2.

Optionally, in one specific embodiment of the preceding embodiment, thecell further comprises a mutation that reduces or eliminates theprotease activity of tsp1.

In certain embodiments that may be combined with the precedingembodiments, the cell has nine protease encoding genes, each of whichcomprise a mutation that reduces or eliminates the correspondingprotease activity, and the nine protease encoding genes are pep1, slp1,gap1, gap2, pep4, pep3, pep5, pep2 and sep1. Optionally, in one specificembodiment of such embodiment, the cell further comprises a mutationthat reduces or eliminates the protease activity of tsp1.

In certain embodiments that may be combined with the precedingembodiments, the cell has ten protease encoding genes, each of whichcomprise a mutation that reduces or eliminates the correspondingprotease activity, and the ten protease encoding genes are pep1, slp1,gap1, gap2, pep4, pep3, pep5, pep2, sep1 and slp8. Optionally, in onespecific embodiment of such embodiment, the cell further comprises amutation that reduces or eliminates the protease activity of tsp1.

In certain embodiments that may be combined with the precedingembodiments, the cell has eleven protease encoding genes, each of whichcomprise a mutation that reduces or eliminates the correspondingprotease activity, and the eleven protease encoding genes are pep1,slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1, slp8 and amp2.Optionally, in one specific embodiment of such embodiment, the cellfurther comprises a mutation that reduces or eliminates the proteaseactivity of tsp1.

In certain embodiments that may be combined with the precedingembodiments, the cell has twelve protease encoding genes, each of whichcomprise a mutation that reduces or eliminates the correspondingprotease activity, and the twelve protease encoding genes are pep1,slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1, slp8, amp2 and slp7.

In certain embodiments that may be combined with the precedingembodiments, the cell has reduced or no protease activity in at leastthirteen proteases, each of the genes encoding the thirteen proteasescomprises a mutation that reduces or eliminates the correspondingprotease activity, and the thirteen proteases are either

-   -   pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1,        slp8, amp2, pep9;    -   pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1,        slp8, amp2, slp7;    -   pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1,        slp8, amp2, slp3.

In certain embodiments that may be combined with the precedingembodiments, the cell has reduced or no protease activity in at leastfourteen proteases, each of the genes encoding the fourteen proteasescomprises a mutation that reduces or eliminates the correspondingprotease activity, and the fourteen proteases are pep1 tsp1 slp1 gap1gap2 pep4 pep3 pep5 pep2 sep1 slp8 amp2 pep9 slp2;

In certain embodiments that may be combined with the precedingembodiments, the cell has reduced or no protease activity in at leastfifteen proteases, each of the genes encoding the fifteen proteasescomprises a mutation that reduces or eliminates the correspondingprotease activity, and the fifteen proteases are either

-   -   pep1 tsp1 slp1 gap1 gap2 pep4 pep3 pep5 pep2 sep1 slp8 amp2 pep9        slp2 mp1; or,    -   pep1 tsp1 slp1 gap1 gap2 pep4 pep3 pep5 pep2 sep1 slp8 amp2 pep9        slp2 mp5.

In certain embodiments that may be combined with the precedentembodiments, the cell has reduced or no protease activity in at leastsixteen, at least seventeen, at least eighteen, at least nineteen, or atleast twenty or more proteases, and said cell comprises at least onemutation that reduces or eliminates the corresponding protease activity,selected from the group consisting of

-   -   an aspartic protease pep6, pep10, pep13, pep14, or pep16;    -   slp like protease slp57433, slp35726, slp60791, or slp109276;    -   gap like protease gap3 or gap4;    -   sedolisin like protease sed2, sed3, or sed5;    -   Group A protease selected from the group of protease65735,        protease77577, protease81087, protease56920, protease122083,        protease79485, protease120998, or protease61127;    -   Group B protease selected from the group of protease21659,        protease58387, protease75159, protease56853, or protease64193;    -   Group C protease selected from the group of protease82452,        protease80762, protease21668, protease81115, protease82141,        protease23475;    -   Group D protease selected from the group of protease121890,        protease22718, protease47127, protease61912, protease80843,        protease66608, protease72612, protease40199; or    -   Group E protease selected from the group of protease22210,        protease111694, protease82577.

In certain embodiments that may be combined with the precedingembodiments, the heterologous polypeptide is a mammalian polypeptide. Incertain embodiments, the mammalian polypeptide is glycosylated.

In certain embodiments, the mammalian polypeptide is selected from animmunoglobulin, an antibody and their antigen-binding fragments, agrowth factor, an interferon, a cytokine, and an interleukin. In certainembodiments, the mammalian polypeptide is an immunoglobulin or anantibody, or their Fc fragment. In certain embodiments, the mammalianpolypeptide is selected from insulin-like growth factor 1 (IGF1), humangrowth hormone (hGH), and interferon alpha 2b (IFNα2b).

In certain embodiments that may be combined with the precedingembodiments, the heterologous polypeptide is a non-mammalianpolypeptide. In certain embodiments, the non-mammalian polypeptide is anaminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,cellulase, chitinase, cutinase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase or xylanase.

In certain embodiments that may be combined with the precedingembodiments, the fungal cell further contains reduced or no detectableactivity of ALG3, a mannosyltransferase enzyme. In certain embodiments,the gene encoding ALG3 contains a mutation that reduces or eliminatesthe corresponding activity. In certain embodiments that may be combinedwith the preceding embodiments, the fungal cell further contains apolynucleotide encoding an α-1,2-mannosidase.

In certain embodiments that may be combined with the precedingembodiments, the fungal cell has a mutation that reduces the expressionof a protease desired to have reduced activity. In certain embodimentsthat may be combined with the preceding embodiments, the mutation is adeletion within the gene encoding the protease. In certain embodimentsthat may be combined with the preceding embodiments, the mutation is adeletion of the portion of the gene encoding the catalytic domain of theprotease. In certain embodiments that may be combined with the precedingembodiments, the fungal cell has a point mutation in the portion of thegene encoding the catalytic domain of the protease.

In other embodiments, the reduction or elimination of protease activityof one or more proteases results from RNAi constructs specific for i)one protease or ii) two or more proteases selected from the groupconsisting of a pep-type protease, a trypsin-like serine protease, agap-type protease, a sedolisin protease and a sip-type protease. Incertain embodiments, RNAi constructs are specific for slp2, slp3, slp5and/or slp6.

In certain embodiments that may be combined with the precedingembodiments, the fungal cell further contains anN-acetylglucosaminyltransferase I catalytic domain and anN-acetylglucosaminyltransferase II catalytic domain. In certainembodiments, the N-acetylglucosaminyltransferase I catalytic domain andthe N-acetylglucosaminyltransferase II catalytic domain are encoded by apolynucleotide. In certain embodiments, theN-acetylglucosaminyltransferase I catalytic domain is encoded by a firstpolynucleotide and the N-acetylglucosaminyltransferase II catalyticdomain is encoded by a second polynucleotide. In certain embodimentsthat may be combined with the preceding embodiments, the fungal cellfurther contains a polynucleotide encoding a mannosidase II and/or agalactosyl transferase. In certain embodiments, the fungal cell containsenzymes selected from the group consisting of α1,2 mannosidase,N-acetylglucosaminyltransferase I, N-acetylglucosaminyltransferase II,mannosidase II and/or galactosyltransferase, said enzymes furthercomprising a targeting peptide, for example a heterologous targetingpeptide for proper localization of the corresponding enzyme. In certainembodiments, the targeting peptide is selected from SEQ ID NOs: 589-594.

In certain embodiments that may be combined with the precedingembodiments, the fungal cell is a Trichoderma fungal cell, aMyceliophthora fungal cell, an Aspergillus fungal cell, a Neurosporafungal cell, a Fusarium or Penicilium fungal cell, or a Chrysosporiumfungal cell. In certain embodiments that may be combined with thepreceding embodiments, the fungal cell is Trichoderma reesei.

In certain embodiments that may be combined with the precedingembodiments, the fungal cell is wild type for pep4 protease. In certainembodiments that may be combined with the preceding embodiments, thefungal cell is wild type for tsp1 protease.

Another aspect includes methods of improving heterologous polypeptidestability, by: a) providing the filamentous fungal cell of any of thepreceding embodiments; and b) culturing the cell such that theheterologous polypeptide is expressed, where the heterologouspolypeptide has increased stability compared to the heterologouspolypeptide produced in a corresponding parental filamentous fungal cellin which the proteases do not have reduced activity, for example, as notcontaining the mutations of the genes encoding the proteases. Anotheraspect includes methods of making a heterologous polypeptide, by: a)providing the filamentous fungal cell of any of the precedingembodiments; b) culturing the host cell such that the heterologouspolypeptide is expressed; and c) purifying the heterologous polypeptide.

In certain embodiments that may be combined with the precedingembodiments, the filamentous fungal cell further contains a carrierprotein. In certain embodiments, the carrier protein is CBH1. In certainembodiments that may be combined with the preceding embodiments, theculturing is in a medium comprising a protease inhibitor. In certainembodiments, the culturing is in a medium having one or two proteaseinhibitors selected from SBTI and chymostatin. In certain embodiments,the protease activities are reduced or eliminated according to theinvention with further co-expression or co-culture such as SBTI, or BBI,e.g. as described in WO2005047302, or slp inhibitor such as pepcinhibitor as described in WO2009071530.

In certain embodiments, the heterologous polypeptide produced accordingto the method is a glycosylated mammalian polypeptide, preferably anantibody or their Fc glycosylated fragments, and at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, or 100% (mole %) of the total N-glycansof the polypeptide consists of Man₃GlcNAc₂ N-glycan or Man5GlcNAc2N-glycan. In other embodiments, the heterologous polypeptide producedaccording to the method is a glycosylated mammalian polypeptide,preferably an antibody or their Fc glycosylated fragments, and at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, or 100% (mole %) of thetotal N-glycans of the polypeptide consists of complex N-glycan, such asG0, G1 or G2 glycoforms or their fucosylated forms, FG0, FG1 and FG2. Incertain embodiments, the heterologous polypeptide produced according tothe method is a glycosylated mammalian polypeptide, preferably anantibody or their Fc glycosylated fragments, and at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, or 100% (mole %) of the total N-glycansof the polypeptide consists of hybrid N-glycan. In certain embodiments,the heterologous polypeptide produced according to the method is aglycosylated mammalian polypeptide, preferably an antibody or their Fcglycosylated fragments, and at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or 100% (mole %) of the total N-glycans of the polypeptideconsists of G1 or G2 N-glycan. Another aspect includes the heterologouspolypeptides, preferably an antibody or their Fc glycosylated fragments,obtainable by the methods as described above.

Another aspect includes Trichoderma fungal cells, or closely relatedspecies, including Myceliophthora fungal cell, Aspergillus fungal cell,Neurospora fungal cell, Penicillium cell, Fusarium cell, orChrysosporium fungal cell, said fungal cell having reduced or nodetectable activity of at least three proteases selected from pep1,pep2, pep3, pep4, pep5, tsp1, slp1, slp2, gap1, and gap2, where the cellfurther contains a recombinant polynucleotide encoding a mammalianpolypeptide produced at a level of at least 2-fold higher than theproduction level of the polypeptide in a corresponding parental fungalcell.

In certain embodiments that may be combined with the precedingembodiments, the Trichoderma or closely related species fungal cell,further contains reduced or no detectable activity of one or more of thefollowing proteases: pep9, amp1, amp2 and sep1.

In certain embodiments, the expression level of the at least threeproteases is reduced or eliminated in the Trichoderma or closely relatedspecies fungal cell. In certain embodiments, genes encoding the at leastthree proteases each comprise a mutation that reduces or eliminates thecorresponding protease activity in the Trichoderma or closely relatedspecies cell. In certain embodiments, the Trichoderma or closely relatedspecies fungal cell includes three protease encoding genes with amutation that reduces or eliminates protease activity, which areselected from gap1, slp1, and pep1. In certain embodiments that may becombined with the preceding embodiments, the mammalian polypeptide inthe Trichoderma or closely related species fungal cell is an antibody,or their antigen-binding fragments, or an immunoglobulin, and the atleast three proteases are selected from pep1, pep3, pep4, tsp1, slp1,slp2, gap1, and gap2. In certain embodiments, the Trichoderma or closelyrelated species fungal cell contains four protease encoding genes, eachof which comprise a mutation that reduces or eliminates thecorresponding protease activity, and the four protease encoding geneswith such mutation are pep1, tsp1, slp1, and gap1. In certainembodiments, the Trichoderma or closely related species fungal cell hasfive protease encoding genes, each of which comprise a mutation thatreduces or eliminates the corresponding protease activity, and the fiveprotease encoding genes with such mutation are pep1, tsp1, slp1, gap1,and pep4. In certain embodiments that may be combined with the precedingembodiments, the mammalian polypeptide in the Trichoderma fungal cell isa growth factor, interferon, cytokine, or interleukin, and the threeproteases with reduced activity are selected from pep1, pep2, pep3,pep4, pep5, pep8, pep11, pep12, gap1, gap2, slp1, slp2, slp7, and,optionally tsp1. In certain embodiments, the Trichoderma or closelyrelated species fungal cell has five protease encoding genes, each ofwhich comprise a mutation that reduces or eliminates the correspondingprotease activity, and the five protease encoding genes with suchmutation are pep1, tsp1, slp1, gap1, and gap2. In certain embodiments,the Trichoderma or closely related species fungal cell has six proteaseencoding genes, each of which comprise a mutation that reduces oreliminates the corresponding protease activity, and the six proteaseencoding genes with such mutation are pep1, tsp1, slp1, gap1, gap2, andpep4. In certain embodiments that may be combined with the precedingembodiments, the Trichoderma or closely related species fungal cell hasseven protease encoding genes, each of which comprise a mutation thatreduces or eliminates the corresponding protease activity, and the sevenprotease encoding genes are pep1, tsp1, slp1, gap1, gap2, pep4, andpep3. In certain embodiments that may be combined with the precedingembodiments, the Trichoderma or closely related species fungal cell haseight protease encoding genes, each of which comprise a mutation thatreduces the corresponding protease activity, and the eight proteaseencoding genes with such mutation are pep1, tsp1, slp1, gap1, gap2,pep4, pep3, and pep5. In certain embodiments that may be combined withthe preceding embodiments, the Trichoderma or closely related speciesfungal cell has eight protease encoding genes, each of which comprise amutation that reduces the corresponding protease activity, and the eightprotease encoding genes with such mutation are selected from either

-   -   (i) pep1, slp1, gap1, gap2, pep4, pep3, pep5, amp1;    -   (ii) pep1, slp1, gap1, gap2, pep4, pep3, pep5, amp2;    -   (iii) pep1, slp1, gap1, gap2, pep4, pep3, pep5, sep1;    -   (iv) pep1, slp1, gap1, gap2, pep4, pep3, pep5, pep9; or,    -   (v) pep1, slp1, gap1, gap2, pep4, pep3, pep5, pep2

In such embodiment, the cell may further comprise an additional mutationthat reduces or eliminates the protease activity of tsp1.

In certain embodiments that may be combined with the precedingembodiments, the Trichoderma or closely related species fungal cell hasnine protease encoding genes, each of which comprise a mutation thatreduces the corresponding protease activity, and the nine proteaseencoding genes with such mutation are pep1, slp1, gap1, gap2, pep4,pep3, pep5, pep2, sep1. In such embodiment, the cell may furthercomprise an additional mutation that reduces or eliminates the proteaseactivity of tsp1.

In certain embodiments that may be combined with the precedingembodiments, the Trichoderma or closely related species fungal cell hasten protease encoding genes, each of which comprise a mutation thatreduces the corresponding protease activity, and the ten proteaseencoding genes with such mutation are pep1, slp1, gap1, gap2, pep4,pep3, pep5, pep2, sep1, slp8. In such embodiment, the cell may furthercomprise an additional mutation that reduces or eliminates the proteaseactivity of tsp1.

In certain embodiments that may be combined with the precedingembodiments, the Trichoderma or closely related species fungal cell haseleven protease encoding genes, each of which comprise a mutation thatreduces the corresponding protease activity, and the eleven proteaseencoding genes with such mutation are pep1, slp1, gap1, gap2, pep4,pep3, pep5, pep2, sep1, slp8, amp2. In such embodiment, the cell mayfurther comprise an additional mutation that reduces or eliminates theprotease activity of tsp1.

In certain embodiments that may be combined with the precedingembodiments, the Trichoderma or closely related species fungal cell hastwelve protease encoding genes, each of which comprise a mutation thatreduces the corresponding protease activity, and the twelve proteaseencoding genes with such mutation are pep1, slp1, gap1, gap2, pep4,pep3, pep5, pep2, sep1, slp8, amp2, slp7.

In certain embodiments that may be combined with the precedingembodiments, the Trichoderma or closely related species fungal cell hasreduced or no protease activity in at least thirteen proteases, each ofthe genes encoding the thirteen proteases comprises a mutation thatreduces or eliminates the corresponding protease activity, and thethirteen proteases are either

-   -   pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1,        slp8, amp2, pep9;    -   pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1,        slp8, amp2, slp7;    -   pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1,        slp8, amp2, slp3.

In certain embodiments that may be combined with the precedingembodiments, the Trichoderma or closely related species fungal cell hasreduced or no protease activity in at least fourteen proteases, each ofthe genes encoding the fourteen proteases comprises a mutation thatreduces or eliminates the corresponding protease activity, and thefourteen proteases are pep1 tsp1 slp1 gap1 gap2 pep4 pep3 pep5 pep2 sep1slp8 amp2 pep9 slp2;

In certain embodiments that may be combined with the precedingembodiments, the Trichoderma or closely related species fungal cell hasreduced or no protease activity in at least fifteen proteases, each ofthe genes encoding the fifteen proteases comprises a mutation thatreduces or eliminates the corresponding protease activity, and thefifteen proteases are either

-   -   pep1 tsp1 slp1 gap1 gap2 pep4 pep3 pep5 pep2 sep1 slp8 amp2 pep9        slp2 mp1; or,    -   pep1 tsp1 slp1 gap1 gap2 pep4 pep3 pep5 pep2 sep1 slp8 amp2 pep9        slp2 mp5.

In certain embodiments that may be combined with the precedingembodiments, the Trichoderma or closely related species fungal cellfurther contains reduced or no detectable activity of one or moreadditional proteases. In certain embodiments, the expression level ofthe one or more additional proteases in the Trichoderma or closelyrelated species fungal cell is reduced or eliminated. In certainembodiments, genes encoding the one or more additional protease in theTrichoderma or closely related species fungal cell each have a mutationthat reduces or eliminates the corresponding protease activity. Incertain embodiments that may be combined with the preceding embodiments,the one or more additional protease encoding genes are selected frompep7, pep8, pep11, pep12, tpp1, gap2, slp3, slp5, slp6, slp7, and slp8.In certain embodiments that may be combined with the precedingembodiments, the one or more additional protease encoding genes areselected from the group consisting of

-   -   an aspartic protease pep6, pep10, pep13, pep14, or pep16;    -   slp like protease slp57433, slp35726, slp60791, or slp109276;    -   gap like protease gap3 or gap4;    -   sedolisin like protease sed2, sed3, or sed5;    -   Group A protease selected from the group of protease65735,        protease77577, protease81087, protease56920, protease122083,        protease79485, protease120998, or protease61127;    -   Group B protease selected from the group of protease21659,        protease58387, protease75159, protease56853, or protease64193;    -   Group C protease selected from the group of protease82452,        protease80762, protease21668, protease81115, protease82141,        protease23475;    -   Group D protease selected from the group of protease121890,        protease22718, protease47127, protease61912, protease80843,        protease66608, protease72612, protease40199; or    -   Group E protease selected from the group of protease22210,        protease111694, protease82577.

In certain embodiments that may be combined with the precedingembodiments, the Trichoderma or closely related species fungal cellfurther contains reduced or no detectable activity of ALG3. In certainembodiments, the gene encoding ALG3 in the Trichoderma or closelyrelated species fungal cell contains a mutation that reduces oreliminates the corresponding activity. In certain embodiments that maybe combined with the preceding embodiments, the Trichoderma or closelyrelated species fungal cell further contains a polynucleotide encodingan α-1,2-mannosidase. In certain embodiments that may be combined withthe preceding embodiments, the mutation reduces or eliminates theexpression of the gene in the Trichoderma or closely related speciesfungal cell. In certain embodiments that may be combined with thepreceding embodiments, the mutation is a deletion of the gene in theTrichoderma or closely related species fungal cell. In certainembodiments that may be combined with the preceding embodiments, themutation is a deletion of the portion of the gene encoding the catalyticdomain of the protease in the Trichoderma or closely related speciesfungal cell. In certain embodiments that may be combined with thepreceding embodiments, the mutation is a point mutation in the portionof the gene encoding the catalytic domain of the protease in theTrichoderma or closely related species fungal cell. In certainembodiments that may be combined with the preceding embodiments, theTrichoderma or closely related species fungal cell further contains aN-acetylglucosaminyltransferase I catalytic domain and anN-acetylglucosaminyltransferase II catalytic domain. In certainembodiments, the N-acetylglucosaminyltransferase I catalytic domain andthe N-acetylglucosaminyltransferase II catalytic domain are encoded by apolynucleotide of the Trichoderma or closely related species fungalcell. In certain embodiments, the N-acetylglucosaminyltransferase Icatalytic domain is encoded by a first polynucleotide and theN-acetylglucosaminyltransferase II catalytic domain is encoded by asecond polynucleotide of the Trichoderma or closely related speciesfungal cell. In certain embodiments that may be combined with thepreceding embodiments, the Trichoderma fungal cell further contains apolynucleotide encoding a mannosidase II. In certain embodiments, theproteases each have at least 70%, at least 75%, at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or 100% sequence identity to an amino acid sequenceselected from SEQ ID NOs: 1, 17, 37, 58, 66, 82, 98, 118, 129, 166, and182. In certain embodiments, the total protease activity in theTrichoderma fungal cell is reduced to 49% or less, 40% or less, 31% orless, 6% or less of the total protease activity of the correspondingTrichoderma parental cell in which the proteases do not have the reducedactivity. In certain embodiments that may be combined with the precedingembodiments, the cell further contains a recombinant polynucleotideencoding a mammalian polypeptide produced at a level of at least 2-foldhigher than the production level of the polypeptide in a correspondingparental Trichoderma fungal cell. In certain embodiments that may becombined with the preceding embodiments, the mammalian polypeptide isproduced in a full length version at a level higher than the productionlevel of the full-length version of the polypeptide in a correspondingparental Trichoderma fungal cell.

Another aspect includes methods of improving heterologous polypeptidestability, by: a) providing the Trichoderma fungal cell of any of thepreceding embodiments; and b) culturing the cell such that theheterologous polypeptide is expressed, where the heterologouspolypeptide has increased stability compared to a host cell notcontaining the mutations of the genes encoding the proteases. Anotheraspect includes methods of making a heterologous polypeptide, e.g. animmunoglobulin or antibody or their glycosylated Fc fragments, by: a)providing the Trichoderma or closely related species fungal cell of anyof the preceding embodiments; b) culturing the host cell such that theheterologous polypeptide is expressed; and c) purifying the heterologouspolypeptide. In certain embodiments that may be combined with thepreceding embodiments, the filamentous fungal cell further contains acarrier protein. In certain embodiments, the carrier protein is CBH1.

DESCRIPTION OF THE FIGURES

FIG. 1 depicts Southern blot analysis showing the generation of the10-fold protease deletion strain M633 (48-70C; M659 is repurified M633).Figure A depicts the expected signal of sep1 ORF: 7.5 kb from M124 (Δ0)and M574 (Δ9), no signal from the transformants. Figure B depicts theexpected signal of sep1 5′ flank: 7.5 kb from M124, 3.1 kb fromtransformants, 3.9 kb from pTTv255. Figure C depicts the expected signalof sep1 3′ flank: 7.5 kb from M124, 3.9 kb from transformants, 3.9 kbfrom pTTv255.

FIG. 2 depicts Southern blot analysis showing the generation of the11-fold protease deletion strain M750 (54-131-2). Figure A depicts theexpected signal of slp8 ORF: 5.6 kb from M124 (Δ0) and M659 (Δ10), nosignal from the transformants. The faint signal pattern seen in allsamples is unspecific background. Figure B depicts the expected signalof slp8 5′ flank: 5.6 kb from M659, 2.9 kb from transformants, 6.7 kbfrom pTTv330. Figure C depicts the expected signal of slp8 3′ flank: 5.6kb from M659, 4.2 kb from transformants, 6.7 kb from pTTv330. StrainM751 (54-159-1) shows multiple signals with both probes indicatingintegration of several deletion cassettes to the genome and putativelyalso genomic rearrangements.

FIG. 3 depicts Southern blot analysis showing the generation of the12-fold protease deletion strain M893 (59-6A). Figure A depicts theexpected signal of amp2 ORF: 5.5 kb from M124 (Δ0) and M750 (Δ11), nosignal from the transformants. Figure B depicts the expected signal ofamp2 5′ flank: 5.5 kb from M124 and M750, 3.6 kb from transformants, 6.6kb from pTTv327. Signal seen at approximately 7 kb for all samples isunspecific background. Figure C depicts the expected signal of amp2 3′flank: 5.5 kb from M124 and M750, 4.5 kb from transformants, 6.6 kb frompTTv327.

FIG. 4 depicts 24 well culture of 9 fold protease deletion strains.Cultures were grown in TrMM with diammonium citrate without ammoniumsulfate, 100 mM PIPPS, 20 g/L spent grain extract, 40 g/L lactose at pH4.5, shaking at 28° C. Immunoblot of interferon alpha 2b expression from0.5 μl culture supernatant.

FIG. 5 depicts a fermentor cultivation of 9 protease deletion strainsexpressing interferon. Strains were grown in TrMM plus 20 g/L yeastextract, 40 g/L cellulose, 80 g/L cellobiose, 40 g/L sorbose, pH 4.5.Immunoblot detecting interferon alpha 2b from 0.1 μl of supernatant.Standard amounts of interferon 50, 100, and 200 ng were used to generatea standard curve.

FIG. 6 depicts an immunoblot showing the interferon alpha 2b productionlevel in the supernatant of the M674 cultivated in the Triab125 andTriab126 fermentations

FIG. 7 depicts a 24 well culture of slp2 silencing strains and controlM577 strain. Cultures were grown in TrMM with diammonium citrate withoutammonium sulfate, 100 mM PIPPS, 20 g/L spent grain extract, 40 g/Llactose at pH 4.5, shaking at 28° C. Immunoblot of interferon alpha 2bexpression from 0.2 μl culture supernatant.

FIG. 8 depicts cultivation of strains M960 and M961 in fermentor usingTrMM in 20 g/L yeast extract, 40 g/L cellulose, 80 g/L cellobiose, and40 g/L sorbose at pH 4.5 with the temperature shifting from 28° to 22°at 48 h. Immunoblot detecting interferon alpha 2b from 0.05 μl ofsupernatant. Interferon standards were 400, 200, 100, 50, and 25 ng.

FIG. 9 depicts a phylogenetic tree of amp1 and amp2 of selectedfilamentous fungi.

FIG. 10 depicts a phylogenetic tree of sep1 of selected filamentousfungi.

FIG. 11 depicts a phylogenetic tree of pep9 of selected filamentousfungi.

FIG. 12 FGF21 production in 24 well cultures treated with proteaseinhibitors. Supernatant samples from day 6 were assayed viaimmunoblotting with a FGF21 standard curve. The primary antibody andreference material was provided by Novartis. The primary antibody wasdiluted to 2 μg/ml in TBST before use. The goat anti-rabbit secondary(Biorad goat anti-rabbit AP #170-6518) was diluted 1:10,000 in TBST.

FIG. 13 Immunoblot detecting FGF21 and CBHI expression from the M393control strain and the new FGF21 transformants from day 4-6. The newtransformants are from M1076 transformation. CBHI carrier was detectedaround 60 kD and the FGF21 free product was at 10 kD.

FIG. 14 Immunoblot using anti-FGF21 antibody to detect expression ofFGF21 from fermentation samples from cultivation of strain M1200 andM1205 with and without inhibitors. Standard amounts of FGF21 areincluded on each blot for quantitation. Supernatants were diluted sothat 0.1 μl was loaded per lane.

FIG. 15 Immunoblot detecting FGF21 expression from the M1200 straingrown in 24 well culture with and without protease inhibitor treatment.Pepstatin, chymostatin, 1,10-phenanthroline were used at a finalconcentration of 10 μM and SBTI was used at 0.1 mg/ml. 2 μl of theculture supernatant plus orange LSB was loaded per well. Wholeanti-FGF21 antibody was generally used to detect all forms of FGF21produced, but in the lower right blot an N-terminal antibody was used todetect the N-terminal containing forms. Also in the lower right blotanti-CBHI antibody was used for detection.

FIG. 16 Immunoblot detecting FGF21 expression from the M1200 straingrown in 24 well culture with and without protease inhibitor treatment.Pepstatin, chymostatin, 1,10-phenanthroline were used at a finalconcentration of 10 μM and SBTI was used at 0.1 mg/ml. 2 μl of theculture supernatant plus orange LSB was loaded per well. Wholeanti-FGF21 antibody and a C-terminal antibody were used to detectspecifically the C-terminus and all forms of the FGF21 protein. In theleft blot anti-CBHI antibody was used for detection.

FIG. 17. Immunoblots showing the MAB01 heavy chain expressed in 24 wellcultures. 0.5 μl of each supernatant was loaded into a 4-15% gel.Detected with anti-IgG heavy chain AP conjugate (Sigma# A3188) diluted1:10,000 in TBST. Developed with Promega AP substrate.

FIG. 18 Immunoblots showing the MAB01 heavy chain. 0.05 μl of eachsupernatant was loaded into a 4-15% gel. The heavy chain was detectedwith an anti-human Fc antibody IRDye 700 DX conjugate (Rockland#609-130-003) diluted 1:30,000 in TBST. The fluorescence at 700 nm wasdetected using an Odyssey CLx near infrared imager (Li-Cor).

FIG. 19 show a phylogenetic tree of a subset of the proteases amenableto deletions.

FIG. 20 graphically depicts normalized protease activity data fromculture supernatants from each of the protease deletion supernatants andthe parent strain M124. Protease activity was measured at pH 5.5 infirst 5 strains and at pH 4.5 in the last three deletion strains.Protease activity is against green fluorescent casein. The six proteasedeletion strain has only 6% of the wild type parent strain and the 7protease deletion strain protease activity was about 40% less than the 6protease deletion strain activity.

DETAILED DESCRIPTION

The present invention relates to improved methods of generatingrecombinant heterologous polypeptides in filamentous fungal cells thathave reduced or no activity of at least three proteases. The presentinvention further relates to improved methods of generating recombinantheterologous polypeptides in filamentous fungal cells that have reducedor no activity in one or more of the following proteases: pep9, amp1,amp2 and sep1. The present invention is based in part upon thesurprising discovery that reducing the activity of a specificcombination of endogenous proteases in filamentous fungal cellsincreases the expression and stability of a variety of recombinantlyexpressed heterologous proteins, such as immunoglobulins and growthfactors. While others have created Trichoderma fungal cells with one ormore proteases inactivated, they have not provided guidance as to whichproteases are most relevant to increasing the expression and stabilityof specific types of proteins, such as mammalian proteins. For example,WO2011/075677 discloses certain proteases that can be knocked out inTrichoderma and even discloses Trichoderma fungal cells that aredeficient in multiple proteases.

However, WO2011/075677 does not provide any guidance regarding which ofthe proteases have an adverse impact on the expression and stability ofmammalian proteins, such as immunoglobulins or growth factors, as noexamples of expression of any mammalian proteins are described therein.Moreover, WO2011/075677 only discloses heterologous expression of asingle fungal protein in each of three different fungal strainsdeficient in a single protease. Thus, one of skill in the art wouldlikely read WO2011/075677 as teaching that inactivating each singleprotease would be sufficient for heterologous protein production. Yoonet al (2009, Appl. Microbiol Biotechnol 82: 691-701, 2010: Appl.Microbiol Biotechnol DOI 10.1007/s00253-010-2937-0) reported theconstruction of quintuple and ten fold protease gene disruptants forheterologous protein production in Aspergillus oryzae. The 10 proteasedisruptant cells improve the production yield of chymosin by only 3.8fold, despite the high number of disrupted protease genes. Van denHombergh et al reported a triple protease gene disruptant of Aspergillusniger. While the data show a reduction in protease activity, there is noexample of any mammalian protein production described herein.WO2002068623 further report Aspergillus niger amp1, sep1 and pep9proteases, and WO2012048334 reports Myceliophtora thermophila amp2, sep1and pep9 proteases. Other reports describe the cloning andcharacterization of sep1 protease in filamentous fungal strains(WO2011077359, WO2009144269, WO200762936 and WO2002045524). The cloningand characterization of pep9 has also been described in WO2012032472,and WO2006110677.

Applicants have surprisingly shown that multiple proteases are relevantto reduction of total protease activity, increasing production ofheterologous proteins and stabilizing the heterologous proteins afterexpression, in filamentous fungal cells, such as Trichoderma fungalcells. In particular, the inventors have identified proteases that areactually expressed in Trichoderma fungal cells (as opposed to merelybeing coded for in the genome) by purifying these proteases anddetermining which have activities that are most relevant in degradingheterologous proteins, such as mammalian proteins. Additionally, theinventors confirmed that deleting the genes responsible for theparticular protease activities achieved a substantial reduction in totalprotease activity, which correlates to an increase in proteinstabilization in terms of both quantity and quality of proteins producedin filamentous fungal cells containing such deletions, and resulted inan increase in the production of full length heterologous proteins inthe cells. It was also found that Trichoderma fungal cells engineered toreduce the activity of at least three protease genes resulted in anunexpected, synergistic increase in the production of full lengthmammalian proteins, such as antibodies, therapeutic protein or antibodyvariants such Fab or single domain antibodies. In other words, theamount of full length mammalian protein produced was greater than thesum of the amounts produced in Trichoderma fungal cells containing onlyone or two protease gene deletions. Thus, in contrast to WO2011/075677,the inventors have shown that production of intact heterologous proteinsin filamentous fungal cells, such as Trichoderma fungal cells, can beachieved by reducing or eliminating the activity of at least threeproteases in the cells.

Accordingly, certain aspects of the present disclosure providefilamentous fungal cells that produce increased levels of a heterologousprotein by having reduced or no activity of at least three proteases,where the cell further contains a recombinant polynucleotide encoding aheterologous polypeptide produced at a level of at least 2-fold higherthan the production level of the polypeptide in a corresponding parentalfilamentous fungal cell in which the proteases do not have the reducedactivity. In other words, the desired increase in the level of theheterologous protein production is determinable by comparing theproduction level of the heterologous protein in a filamentous fungalcell having the reduced activity of at least three proteases, to that ofa filamentous fungal cell which does not have such reduced activity, butis otherwise identical to the cell exhibiting the increased level.

Other aspects of the present disclosure provide methods of improvingheterologous polypeptide stability, by: a) providing a filamentousfungal cell of the present disclosure having reduced or no activity ofat least three proteases, where the cell further contains a recombinantpolynucleotide encoding a heterologous polypeptide; and b) culturing thecell such that the heterologous polypeptide is expressed, where theheterologous polypeptide has increased stability compared to a host cellnot containing the mutations of the genes encoding the proteases.

Still other aspects of the present disclosure provide methods of makinga heterologous polypeptide, by: a) providing a filamentous fungal cellof the present disclosure having reduced or no activity of at leastthree proteases, where the cell further contains a recombinantpolynucleotide encoding a heterologous polypeptide; b) culturing thehost cell such that the heterologous polypeptide is expressed; and c)purifying the heterologous polypeptide.

Certain aspects of the present disclosure also provide Trichodermafungal cells that produce increased levels of a mammalian polypeptide byhaving reduced or no activity of at least three proteases selected frompep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1, slp1, slp2,gap1, and gap2, where the cell further contains a recombinantpolynucleotide encoding a mammalian polypeptide produced at a level ofat least 2-fold higher than the production level of the polypeptide in acorresponding parental Trichoderma fungal cell in which the proteases donot have the reduced activity. In other words, the desired increase inthe level of the heterologous protein production is determinable bycomparing the production level of the heterologous protein in aTrichoderma fungal cell having the reduced activity of at least threeproteases, to that of a Trichoderma fungal cell which does not have suchreduced activity, but is otherwise identical to the cell exhibiting theincreased level.

Certain aspects of the present disclosure also provide Trichodermafungal cells that produce increased levels of a mammalian polypeptide byhaving reduced or no activity of at least one or more proteases selectedfrom pep9, amp1, amp2, mp1, mp2, mp3, mp4, mp5 and sep1.

Other aspects of the present disclosure provide methods of improvingmammalian polypeptide stability, by: a) providing a Trichoderma fungalcell of the present disclosure having reduced activity of at least threeproteases, where the cell further contains a recombinant polynucleotideencoding a mammalian polypeptide; and b) culturing the cell such thatthe mammalian polypeptide is expressed, where the mammalian polypeptidehas increased stability compared to a host cell not containing themutations of the genes encoding the proteases.

Other aspects of the present disclosure provide methods of improvingmammalian polypeptide stability, by: a) providing a Trichoderma fungalcell of the present disclosure having reduced activity of at least oneor more proteases selected from pep9, amp1, amp2, mp1, mp2, mp3, mp4,mp5 and sep1, where the cell further contains a recombinantpolynucleotide encoding a mammalian polypeptide; and b) culturing thecell such that the mammalian polypeptide is expressed, where themammalian polypeptide has increased stability compared to a host cellnot containing the mutations of the genes encoding the proteases.

Further aspects of the present disclosure provide methods of making amammalian polypeptide, by: a) providing a Trichoderma fungal cell of thepresent disclosure having reduced activity of at least three protease,where the cell further contains a recombinant polynucleotide encoding amammalian polypeptide; b) culturing the host cell such that themammalian polypeptide is expressed; and c) purifying the mammalianpolypeptide.

Definitions

As used herein, an “immunoglobulin” refers to a multimeric proteincontaining a heavy chain and a light chain covalently coupled togetherand capable of specifically combining with antigen. Immunoglobulinmolecules are a large family of molecules that include several types ofmolecules such as IgM, IgD, IgG, IgA, and IgE.

As used herein, an “antibody” refers to intact immunoglobulin molecules,as well as fragments thereof which are capable of binding an antigen.These include hybrid (chimeric) antibody molecules (see, e.g., Winter etal. Nature 349:293-99225, 1991; and U.S. Pat. No. 4,816,567 226);F(ab′)2 and F(ab) fragments and Fv molecules; non-covalent heterodimers[227, 228]; single-chain Fv molecules (scFv) (see, e.g., Huston et al.Proc. Natl. Acad. Sci. U.S.A. 85:5897-83, 1988); dimeric and trimericantibody fragment constructs; minibodies (see, e.g., Pack et al. Biochem31, 1579-84, 1992; and Cumber et al. J. Immunology 149B, 120-26, 1992);humanized antibody molecules (see e.g., Riechmann et al. Nature 332,323-27, 1988; Verhoeyan et al. Science 239, 1534-36, 1988; and GB2,276,169); and any functional fragments obtained from such molecules,as well as antibodies obtained through non-conventional processes suchas phage display. Preferably, the antibodies are monoclonal antibodies.Methods of obtaining monoclonal antibodies are well known in the art.

As used herein, a “peptide” and a “polypeptide” are amino acid sequencesincluding a plurality of consecutive polymerized amino acid residues.For purpose of this invention, typically, peptides are those moleculesincluding up to 50 amino acid residues, and polypeptides include morethan 50 amino acid residues. The peptide or polypeptide may includemodified amino acid residues, naturally occurring amino acid residuesnot encoded by a codon, and non-naturally occurring amino acid residues.As used herein, “protein” may refer to a peptide or a polypeptide of anysize.

Proteases of the Invention

The invention described herein relates to filamentous fungal cells, suchas Trichoderma fungal cells, that produce increased levels of aheterologous polypeptide, such as a mammalian polypeptide, by havingreduced or no detectable activity of at least three proteases found inthe cells. Such proteases found in filamentous fungal cells that expressa heterologous polypeptide normally catalyze significant degradation ofthe expressed recombinant polypeptides. Thus, by reducing or eliminatingthe activity of proteases in filamentous fungal cells that express aheterologous polypeptide, the stability of the expressed polypeptide isincreased, resulting in an increased level of production of thepolypeptide, and in some circumstances, improved quality of the producedpolypeptide (e.g., full-length instead of degraded).

Proteases including, without limitation, aspartic proteases,trypsin-like serine proteases, subtilisin proteases, glutamic proteases,metalloproteases and sedolisin proteases. Such proteases may beidentified and isolated from filamentous fungal cells and tested todetermine whether reduction in their activity affects the production ofa recombinant polypeptide from the filamentous fungal cell. Methods foridentifying and isolating proteases are well known in the art, andinclude, without limitation, affinity chromatography, zymogram assays,and gel electrophoresis. An identified protease may then be tested bydeleting the gene encoding the identified protease from a filamentousfungal cell that expresses a recombinant polypeptide, such aheterologous or mammalian polypeptide, and determining whether thedeletion results in a decrease in total protease activity of the cell,for example, to a level of 49% or less, or 31% or less, of the totalprotease activity of the corresponding parental filamentous fungal cell;and an increase in the level of production of the expressed recombinantpolypeptide, for example two-fold higher than the production level inthe corresponding parental filamentous fungal cell. Methods for deletinggenes, measuring total protease activity, and measuring levels ofproduced protein are well known in the art and include the methodsdescribed herein. The “corresponding parental filamentous fungal cell”refers to the corresponding cell in which the proteases do not havereduced or eliminated activity.

Aspartic Proteases

Aspartic proteases are enzymes that use an aspartate residue forhydrolysis of the peptide bonds in polypeptides and proteins. Typically,aspartic proteases contain two highly-conserved aspartate residues intheir active site which are optimally active at acidic pH. Asparticproteases from eukaryotic organisms such as Trichoderma fungi includepepsins, cathepsins, and renins. Such aspartic proteases have atwo-domain structure, which is thought to arise from an ancestral geneduplication. Consistent with such a duplication event, the overall foldof each domain is similar, though the sequences of the two domains havebegun to diverge. Each domain contributes one of the catalytic aspartateresidues. The active site is in a cleft formed by the two domains of theaspartic proteases. Eukaryotic aspartic proteases further includeconserved disulfide bridges, which can assist in identification of thepolypeptides as being aspartic acid proteases.

Fifteen aspartic proteases have been identified in Trichoderma fungalcells: pep1 (tre74156), pep2 (tre53961), pep3 (tre121133), pep4(tre77579), pep5 (tre81004), pep6 (tre68662), pep7 (tre58669), pep8(tre122076), pep9 (tre79807), pep10 (tre78639), pep11 (tre121306), pep12(tre119876), pep13 (tre76887), pep14 (tre108686) and pep16 (tre110490).

Pep1

Examples of suitable pep1 proteases include, without limitation,Trichoderma reesei pep1 (SEQ ID NO: 1), Hypocrea lixii gi|11558498 (SEQID NO: 2), Trichoderma asperellum gi|47027997 (SEQ ID NO: 3),Trichoderma atroviride jgi|Triat2|297887 (SEQ ID NO: 4), Trichodermavirens jgi|TriviGv29_8_2|81777 (SEQ ID NO: 5), Aspergillus fumigatusjgi|Trire2|afm:Afu5g13300 (SEQ ID NO: 6), Aspergillus oryzae gi|94730408(SEQ ID NO: 7), Metarhizium anisopliae gi|322712783 (SEQ ID NO: 8),Gibberella zeae gi|46126795 (SEQ ID NO: 9), Fusarium venenatumgi|18448713 (SEQ ID NO: 10), Fusarium oxysporum gi|342879173 (SEQ ID NO:11), Grosmannia clavigera gi|320591399 (SEQ ID NO: 12), Verticilliumalboatrum gi|302422750 (SEQ ID NO: 13), Chaetomium globosum gi|116182964(SEQ ID NO: 14), Neurospora crassa gi|85110723 (SEQ ID NO: 15),Neurospora tetrasperma gi|336463990 (SEQ ID NO: 16), Myceliophthorathermophila gi367030924 (SEQ ID NO: 491), Penicillium chrysogenumgi255953325 (SEQ ID NO: 492), Aspergillus niger gi350639535 (SEQ ID NO:493), Aspergillus nidulans gi67541436 (SEQ ID NO: 494), and homologsthereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a pep1 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 1-16, SEQ ID NOs:491-494. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 1-16, SEQ ID NOs:491-494.

In some embodiments, pep1 is T. reesei pep1. The amino acid sequenceencoded by T. reesei pep1 is set forth in SEQ ID NO: 1. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 1. In further embodiments, the protease has 100% identity to SEQID NO: 1.

Pep2

Examples of suitable pep2 proteases include, without limitation,Trichoderma reesei pep2 (SEQ ID NO: 182), T atroviride jgi|Triat2|142040(SEQ ID NO: 183), T virens jgi|TriviGv29_8_2|53481 (SEQ ID NO: 184),Cordyceps militaris CM01 gi|346326575 (SEQ ID NO: 185), Neurosporacrassa gi 85111370 (SEQ ID NO: 495), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a pep2 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 182-185, SEQ ID NO:495. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 182-185, SEQ ID NO:495.

In some embodiments, pep2 is T. reesei pep2. The amino acid sequenceencoded by T. reesei pep2 is set forth in SEQ ID NO: 182. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 182. In further embodiments, the protease has 100% identity toSEQ ID NO: 182.

Pep3

Examples of suitable pep3 proteases include, without limitation,Trichoderma reesei pep3 (SEQ ID NO: 17), T atroviride jgi|Triat2 (SEQ IDNO: 18), T virens, jgi|TriviGv29_8_2 (SEQ ID NO: 19), Hypocrea lixiigi|145583125 (SEQ ID NO: 20), Trichoderma asperellum gi|51860175 (SEQ IDNO: 21), Aspergillus niger gi|317025164 (SEQ ID NO: 22), Aspergillusfumigatus gi|159122534 (SEQ ID NO: 23), Aspergillus niger gi|134054572(SEQ ID NO: 24), Cordyceps militaris, gi|346318620 (SEQ ID NO: 25),Glomerella graminicola gi|310800156 (SEQ ID NO: 26), Fusarium oxysporumgi|342871221 (SEQ ID NO: 27), Grosmannia clavigera gi|320591121 (SEQ IDNO: 28), Botryotinia fuckeliana gi|12002205 (SEQ ID NO: 29), Thielaviaterrestris gi|346997107 (SEQ ID NO: 30), Sclerotinia sclerotiorumgi|156055954 (SEQ ID NO: 31), Chaetomium globosum gi|116197829 (SEQ IDNO: 32), Neurospora tetrasperma gi|336472132 (SEQ ID NO: 33), Neurosporacrassa gi|85102020 (SEQ ID NO: 34), Neosartorya fischeri gi|119467426(SEQ ID NO: 35), Penicillium marneffei gi|212534792 (SEQ ID NO: 36), M.thermophila gi367025909 (SEQ ID NO: 496), P. chrysogenum gi255947264(SEQ ID NO: 497), A. oryzae 391870123 (SEQ ID NO: 498), and homologsthereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a pep3 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 17-36, SEQ ID NOs:496-498. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 17-36, SEQ ID NOs: 496-498.

In some embodiments, pep3 is T. reesei pep3. The amino acid sequenceencoded by T. reesei pep3 is set forth in SEQ ID NO: 17. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 17. In further embodiments, the protease has 100% identity to SEQID NO: 17.

Pep4

Examples of suitable pep4 proteases include, without limitation,Trichoderma reesei pep4 (SEQ ID NO: 37), T virens jgi|TriviGv29_8_2 (SEQID NO: 38), T atroviride jgi|Triat2 (SEQ ID NO: 39), Trichodermaaureoviride gi|193735605 (SEQ ID NO: 40), Aspergillus niger gi|145232965(SEQ ID NO: 41), Aspergillus fumigatus gi|70999520 (SEQ ID NO: 42),Aspergillus clavatus gi|121705756 (SEQ ID NO: 43), Nectria haematococcagi|302899226 (SEQ ID NO: 44), Glomerella graminicola gi|310796316 (SEQID NO: 45), Cordyceps militaris gi|346322842 (SEQ ID NO: 46), Gibberellazeae gi|46138535 (SEQ ID NO: 47), Metarhizium anisopliae gi|322708430(SEQ ID NO: 48), Fusarium oxysporum gi|342882947 (SEQ ID NO: 49),Metarhizium acridum gi|322700747 (SEQ ID NO: 50), Verticillium dahliae,gi|346973691 (SEQ ID NO: 51), Botryotinia fuckeliana gi|154309857 (SEQID NO: 52), Chaetomium globosum gi|116203505 (SEQ ID NO: 53), Thielaviaterrestris gi|347001590 (SEQ ID NO: 54), Magnaporthe oryzae gi|39973863(SEQ ID NO: 55), Tuber melanosporum gi|296417651 (SEQ ID NO: 56),Neurospora crassa gi|85094599 (SEQ ID NO: 57), M. thermophilagi367031892 gi255947264 (SEQ ID NO: 499), P. chrysogenum gi255936729gi255947264 (SEQ ID NO: 500), A. oryzae gi169770745 gi255947264 (SEQ IDNO: 501), A. nidulans gi67524891 gi255947264 (SEQ ID NO: 502), andhomologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a pep4 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 37-57, SEQ ID NOs:499-502. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 37-57, SEQ ID NOs:499-502.

In some embodiments, pep4 is T. reesei pep4. The amino acid sequenceencoded by T. reesei pep4 is set forth in SEQ ID NO: 37. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 37. In further embodiments, the protease has 100% identity to SEQID NO: 37.

Pep5

Examples of suitable pep5 genes include, without limitation, Trichodermareesei pep5 (SEQ ID NO: 58), T virens jgi|TriviGv29_8_2 (SEQ ID NO: 59),T atroviride jgi|Triat2|277859 (SEQ ID NO: 60), Metarhizium acridumgi|322695806 (SEQ ID NO: 61), Fusarium oxysporum gi|156071418 (SEQ IDNO: 62), Cordyceps militaris gi|346324830 (SEQ ID NO: 63), Gibberellazeae gi|46124247 (SEQ ID NO: 64), Verticillium dahliae gi|346978752 (SEQID NO: 65), M. thermophila gi367019798 (SEQ ID NO: 503), and homologsthereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a pep5 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 58-65, SEQ ID NO:503. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 58-65, SEQ ID NO:503.

In some embodiments, pep5 is T. reesei pep5. The amino acid sequenceencoded by T. reesei pep5 is set forth in SEQ ID NO: 58. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 58. In further embodiments, the protease has 100% identity to SEQID NO: 58.

Pep7

Examples of suitable pep7 genes include, without limitation, Trichodermareesei pep7 (SEQ ID NO: 186), Trichoderma atroviride jgi|Triat2 (SEQ IDNO: 187), Trichoderma virens jgi|TriviGv29_8_2 (SEQ ID NO: 188),Glomerella graminicola gi|310800487 (SEQ ID NO: 189), Metarhiziumacridum gi|322700577 (SEQ ID NO: 190), Thielavia terrestris gi|347003264(SEQ ID NO: 191), Podospora anserine gi|171680938 (SEQ ID NO: 192),Chaetomium thermophilum gi|340905460 (SEQ ID NO: 193), Verticilliumdahliae gi|346975960 (SEQ ID NO: 194), Myceliophthora thermophilagi|347009870, gi367026634 (SEQ ID NO: 195), Neurospora crassagi|85090078 (SEQ ID NO: 196), Magnaporthe oryzae gi|39948622 (SEQ ID NO:197), Chaetomium globosum gi|116191517 (SEQ ID NO: 198), Magnaportheoryzae gi|39970765 (SEQ ID NO: 199), A. nidulans gi67522232 (SEQ ID NO:504), A. niger gi350630464 (SEQ ID NO: 505), A. oryzae gi317138074 (SEQID NO: 506), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a pep7 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 186-199, SEQ ID NOs:504-506. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 186-199, SEQ ID NOs:504-506.

In some embodiments, pep7 is T. reesei pep7. The amino acid sequenceencoded by T. reesei pep7 is set forth in SEQ ID NO: 186. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 186. In further embodiments, the protease has 100% identity toSEQ ID NO: 186.

Pep8

Examples of suitable pep8 genes include, without limitation, Trichodermareesei pep8 EGR48424 (SEQ ID NO: 507), Trichoderma virens EHK19238 (SEQID NO: 508), Trichoderma atroviride EHK40047 (SEQ ID NO: 509),Neurospora tetrasperma EGO53367 (SEQ ID NO: 510), Myceliophthorathermophila XP_003658897 (SEQ ID NO: 511), Neurospora crassa XP_965343(SEQ ID NO: 512), Metarhizium anisopliae EFZ03501 (SEQ ID NO: 513),Thielavia terrestris XP_003656869 (SEQ ID NO: 514), Fusarium oxysporumEGU79769 (SEQ ID NO: 515), and Gibberella zeae XP_381566 (SEQ ID NO:516), Magnaporthe oryzae XP_° ° 3714540.1 (SEQ ID NO:517), P.chrysogenum XP_002557331 (SEQ ID NO: 518), A. oryzae XP_001822899.1 (SEQID NO: 519), A. nidulans XP_664091.1 (SEQ ID NO: 520), A. nigerEHA24387.1 (SEQ ID NO: 521), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a pep8 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 507-521. In some embodiments, theprotease has 100% identity to an amino acid sequence selected from SEQID NOs: 507-521.

In some embodiments, pep8 is T. reesei pep8. The amino acid sequenceencoded by T. reesei pep8 is set forth in SEQ ID NO: 507. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 507. In further embodiments, the protease has 100% identity toSEQ ID NO: 507.

Pep9

Examples of suitable pep9 genes include, without limitation, Trichodermareesei pep9 79807 (SEQ ID NO: 750), Trichoderma virens 158334 (SEQ IDNO: 751), Trichoderma atroviride 90832 (SEQ ID NO: 752), Fusariumgraminicola XP_384573.1 (SEQ ID NO: 753), Neurospora crassaXP_001727974.1 (SEQ ID NO: 754), Myceliophthora thermophilaXP_003667167.1 (SEQ ID NO: 528), Aspergillus oryzae XP_001821372.2 (SEQID NO: 529), Aspergillus niger ABM05950.1 (SEQ ID NO: 755), Aspergillusfumigatus XP_752122.1 (SEQ ID NO: 756), Aspergillus nidulans XP_662026.1(SEQ ID NO: 757), Penicillium Wisconsin XP_002565726.1 (SEQ ID NO: 758)and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a pep9 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 750-758. In some embodiments, theprotease has 100% identity to an amino acid sequence selected from SEQID NOs: 750-758.

In some embodiments, pep9 is T. reesei pep9. The amino acid sequenceencoded by T. reesei pep9 is set forth in SEQ ID NO: 750. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 750. In further embodiments, the protease has 100% identity toSEQ ID NO: 750.

Pep11

Examples of suitable pep11 genes include, without limitation,Trichoderma reesei pep11 EGR49498 (SEQ ID NO: 522), Trichoderma virensEHK26120 (SEQ ID NO: 523), Trichoderma atroviride EHK41756 (SEQ ID NO:524), Fusarium pseudograminearum EKJ74550 (SEQ ID NO: 525), Metarhiziumacridum EFY91821 (SEQ ID NO: 526), and Gibberella zeae XP_384151 (SEQ IDNO: 527), M. thermophila XP_003667387.1 (SEQ ID NO: 528), N. crassaXP_960328.1 (SEQ ID NO: 529), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a pep11 protease, has an amino acid sequencehaving 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an aminoacid sequence selected from SEQ ID NOs: 522-529. In some embodiments,the protease has 100% identity to an amino acid sequence selected fromSEQ ID NOs: 522-529.

In some embodiments, pep11 is T. reesei pep11. The amino acid sequenceencoded by T. reesei pep11 is set forth in SEQ ID NO: 522. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 522. In further embodiments, the protease has 100% identity toSEQ ID NO: 522.

Pep12

Examples of suitable pep12 genes include, without limitation,Trichoderma reesei pep12 EGR52517 (SEQ ID NO: 530), Trichoderma virenspep12 EHK18859 (SEQ ID NO: 531), Trichoderma atroviride pep12 EHK45753(SEQ ID NO: 532), Fusarium pseudograminearum pep12 EKJ73392 (SEQ ID NO:533), Gibberella zeae pep12 XP_388759 (SEQ ID NO: 534), and Metarhiziumanisopliae pep12 EFY95489 (SEQ ID NO: 535), N. crassa XP_964574.1 (SEQID NO: 536), M. thermophila XP_003659978.1 (SEQ ID NO: 537), andhomologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a pep12 protease, has an amino acid sequencehaving 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an aminoacid sequence selected from SEQ ID NOs: 530-537. In some embodiments,the protease has 100% identity to an amino acid sequence selected fromSEQ ID NOs: 530-537.

In some embodiments, pep12 is T. reesei pep12. The amino acid sequenceencoded by T. reesei pep12 is set forth in SEQ ID NO: 530. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 530. In further embodiments, the protease has 100% identity toSEQ ID NO: 530.

Pep6, pep10, pep13, pep14 and pep16

Other aspartic proteases include, without limitation, T. reeseipep6_(Tre68662, SEQ ID NO:880), pep10_(Tre78639, SEQ ID NO:881),pep13_(Tre76887, SEQ ID NO:882), pep14_(Tre108686, SEQ ID NO:883), orpep16_(Tre110490, SEQ ID NO:884), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a pep6, pep10, pep13, pep14, pep16 protease, hasan amino acid sequence having 50% or more identity (e.g. 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%or more to an amino acid sequence selected from SEQ ID NOs: 880-884. Insome embodiments, the protease has 100% identity to an amino acidsequence selected from SEQ ID NOs: 880-884.

Trypsin-Like Serine Proteases

Trypsin-like serine proteases are enzymes with substrate specificitysimilar to that of trypsin. Trypsin-like serine proteases use a serineresidue for hydrolysis of the peptide bonds in polypeptides andproteins. Typically, trypsin-like serine proteases cleave peptide bondsfollowing a positively-charged amino acid residue. Trypsin-like serineproteases from eukaryotic organisms such as Trichoderma fungi includetrypsin 1, trypsin 2, and mesotrypsin. Such trypsin-like serineproteases generally contain a catalytic triad of three amino acidresidues (such as histidine, aspartate, and serine) that form a chargerelay that serves to make the active site serine nucleophilic.Eukaryotic trypsin-like serine proteases further include an “oxyanionhole” formed by the backbone amide hydrogen atoms of glycine and serine,which can assist in identification of the polypeptides as beingtrypsin-like serine proteases.

One trypsin-like serine protease has been identified in Trichodermafungal cells: tsp1 (tre73897). As discussed below, tsp1 has beendemonstrated to have a significant impact on expression of recombinantpolypeptides, such as immunoglobulins.

As discussed in Example 3 of WO 2013/102674, serine proteases werepurified from Trichoderma and shown to have multiple protease activitiesthat degrade mammalian proteins. Of these activities, tsp1 wasidentified as a trypsin-like serine protease. The tsp1 protease gene wasthen deleted from Trichoderma fungal cells and it was demonstrated thatdeleting tsp1 achieved a significant reduction in total proteaseactivity resulting in increased stabilization of mammalian proteinsproduced by the cells.

Examples of suitable tsp1 proteases include, without limitation,Trichoderma reesei tsp1 (SEQ ID NO: 66), Trichoderma atroviridejgi|Triat2|298187 (SEQ ID NO: 67), jgi|TriviGv29_8_2 (SEQ ID NO: 68),Hypocrea lixii gi|145583579 (SEQ ID NO: 69), Hypocrea lixii gi|63025000(SEQ ID NO: 70), Sclerotinia sclerotiorum gi|156052735 (SEQ ID NO: 71),Botryotinia fuckeliana gi|154314937 (SEQ ID NO: 72), Phaeosphaerianodorum gi|169605891 (SEQ ID NO: 73), Leptosphaeria maculansgi|312219044 (SEQ ID NO: 74), Verticillium dahliae gi|37992773 (SEQ IDNO: 75), Cochliobolus carbonum gi|1072114 (SEQ ID NO: 76), Metarhiziumacridum gi|322695345 (SEQ ID NO: 77), Metarhizium anisopliae gi|4768909(SEQ ID NO: 78), gi|464963 (SEQ ID NO: 79), Gibberella zeae gi|46139299(SEQ ID NO: 80), Metarhizium anisopliae (SEQ ID NO: 81), A. nidulansgi67523821 (SEQ ID NO: 538) and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically tsp1 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 66-81, SEQ ID NO:538. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 66-81, SEQ ID NO:538.

In some embodiments, tsp1 is T. reesei tsp1. The amino acid sequenceencoded by T. reesei tsp1 is set forth in SEQ ID NO: 66. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 66. In further embodiments, the protease has 100% identity to SEQID NO: 66.

Subtilisin Proteases

Subtilisin proteases are enzymes with substrate specificity similar tothat of subtilisin. Subtilisin proteases use a serine residue forhydrolysis of the peptide bonds in polypeptides and proteins. Generally,subtilisin proteases are serine proteases that contain a catalytic triadof the three amino acids aspartate, histidine, and serine. Thearrangement of these catalytic residues is shared with the prototypicalsubtilisin from Bacillus licheniformis. Subtilisin proteases fromeukaryotic organisms such as Trichoderma fungi include furin, MBTPS1,and TPP2. Eukaryotic trypsin-like serine proteases further include anaspartic acid residue in the oxyanion hole. Subtilisin protease slp7resembles also sedolisin protease tpp1.

Seven subtilisin proteases have been identified in Trichoderma fungalcells: slp1 (tre51365); slp2 (tre123244); slp3 (tre123234); slp5(tre64719), slp6 (tre121495), slp7 (tre123865), and slp8 (tre58698).

Slp1

Examples of suitable slp1 proteases include, without limitation,Trichoderma reesei slp1 (SEQ ID NO: 82), Trichoderma atroviridejgi|Triat2 (SEQ ID NO: 83), Trichoderma atroviride jgi|Triat2 (SEQ IDNO: 84), Trichoderma virens jgi|TriviGv29_8_2 (SEQ ID NO: 85), Hypocrealixii gi|145583581 (SEQ ID NO: 86), Metarhizium acridum gi|322694632(SEQ ID NO: 87), Fusarium oxysporum gi|342877080 (SEQ ID NO: 88),Gibberella zeae gi|46139915 (SEQ ID NO: 89), Epichloe festucaegi|170674476 (SEQ ID NO: 90), Nectria haematococca gi|302893164 (SEQ IDNO: 91), Sordaria macrospore gi|336266150 (SEQ ID NO: 92), Glomerellagraminicola gi|310797947 (SEQ ID NO: 93), Neurospora tetraspermagi|336469805 (SEQ ID NO: 94), Neurospora crassa gi|85086707 (SEQ ID NO:95), Magnaporthe oryzae gi|145608997 (SEQ ID NO: 96), Chaetomiumglobosum gi|116208730 (SEQ ID NO: 97), M. thermophila gi367029081 (SEQID NO: 539), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a slp1 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 82-97, SEQ ID NO:539. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 82-97, SEQ ID NO:539.

In some embodiments, slp1 is T. reesei slp1. The amino acid sequenceencoded by T. reesei slp1 is set forth in SEQ ID NO: 82. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 82. In further embodiments, the protease has 100% identity to SEQID NO: 82.

Slp2

Examples of suitable slp2 proteases include, without limitation,Trichoderma reesei slp2 (SEQ ID NO: 98), T atroviride jgi|Triat2 (SEQ IDNO: 99), T virens jgi|TriviGv29_8_2 (SEQ ID NO: 100), Hypocrea lixiigi|115111226 (SEQ ID NO: 101), Aspergillus fumigatus gi|70997972 (SEQ IDNO: 102), Nectria haematococca gi|302915240 (SEQ ID NO: 103), Gibberellazeae gi|46105128 (SEQ ID NO: 104), Isaria farinose gi|68165000 (SEQ IDNO: 105), Glomerella graminicola gi|310797854 (SEQ ID NO: 106), Epichloefestucae gi|170674491 (SEQ ID NO: 107), Metarhizium acridum gi|322697754(SEQ ID NO: 108), Acremonium sp. F11177 gi|147225254 (SEQ ID NO: 109),Ophiostoma piliferum gi|15808807 (SEQ ID NO: 110), Neurosporatetrasperma gi|336463649 (SEQ ID NO: 111), Chaetomium thermophilumgi|340992600 (SEQ ID NO: 112), Metarhizium flavoviride gi|254351265 (SEQID NO: 113), Podospora anserine gi|171680111 (SEQ ID NO: 114),Magnaporthe oryzae gi|39943180 (SEQ ID NO: 115), Sclerotiniasclerotiorum gi|156058540 (SEQ ID NO: 116), Talaromyces stipitatusgi|242790441 (SEQ ID NO: 117), M. thermophila gi367021472 (SEQ ID NO:540), A. niger gi|45237646 (SEQ ID NO: 541), A. oryzae gi|69780712 (SEQID NO: 542), P. chrysogenum gi255955889 (SEQ ID NO: 543), A. nidulansgi259489544 (SEQ ID NO: 544), N. crassa gi85084841 (SEQ ID NO: 545), andhomologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a slp2 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 98-117, SEQ ID NOs:540-545. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 98-117, SEQ ID NOs:540-545.

In some embodiments, slp2 is T. reesei slp2. The amino acid sequenceencoded by T. reesei slp2 is set forth in SEQ ID NO: 98. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 98. In further embodiments, the protease has 100% identity to SEQID NO: 98.

Slp3

Examples of suitable slp3 proteases include, without limitation,Trichoderma reesei slp2 (SEQ ID NO: 166), T. atroviride jgi|Triat2 (SEQID NO: 167), T. virens jgi|TriviGv29_8_2 (SEQ ID NO: 168), Hypocreakoningii gi|124295071 (SEQ ID NO: 169), Purpureocillium lilacinumgi|130750164 (SEQ ID NO: 170), Metarhizium anisopliae gi|16215677 (SEQID NO: 171), Hirsutella rhossiliensis gi|90655148 (SEQ ID NO: 172),Tolypocladium inflatum gi|18542429 (SEQ ID NO: 173), Metacordycepschlamydosporia gi|19171215 (SEQ ID NO: 174), Cordyceps militarisgi|346321368 (SEQ ID NO: 175), Fusarium sp. gi|628051 (SEQ ID NO: 176),Neurospora tetrasperma gi|336471881 (SEQ ID NO: 177), Chaetomiumglobosum gi|116197403 (SEQ ID NO: 178), Neurospora crassa gi|85084841(SEQ ID NO: 179), Fusarium oxysporum gi|56201265 (SEQ ID NO: 180),Gibberella zeae gi|46114268 (SEQ ID NO: 181), M. thermophila gi367026259(SEQ ID NO: 546), A. nidulans gi67538776 (SEQ ID NO: 547), A. oryzaegi169771349 (SEQ ID NO: 222), A. niger gi470729 (SEQ ID NO: 223), andhomologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a slp3 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 166-181, SEQ ID NOs:546-547, SEQ IDNOs:222-223. In some embodiments, the protease has 100% identity to anamino acid sequence selected from SEQ ID NOs: 166-181, SEQ IDNOs:546-547, SEQ ID NOs:222-223.

In some embodiments, slp3 is T. reesei slp3. The amino acid sequenceencoded by T. reesei slp3 is set forth in SEQ ID NO: 166. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 166. In further embodiments, the protease has 100% identity toSEQ ID NO: 166.

Slp5

Examples of suitable slp5 proteases include, without limitation,Trichoderma reesei slp5 (SEQ ID NO: 200), T. atroviride jgi|Triat2 (SEQID NO: 201), T. virens jgi|TriviGv29_8_2 (SEQ ID NO: 202), Hypocrealixii gi|118161442 (SEQ ID NO: 203), Fusarium oxysporum gi|342883549(SEQ ID NO: 204), Gibberella zeae gi|46135733 (SEQ ID NO: 205),Glomerella graminicola gi|310796396 (SEQ ID NO: 206), Nectriahaematococca gi|302927954 (SEQ ID NO: 207), Cordyceps militarisgi|346319783 (SEQ ID NO: 208), Neurospora crassa gi|85094084 (SEQ ID NO:209), Neurospora tetrasperma gi|336467281 (SEQ ID NO: 210), Verticilliumdahliae gi|346971706 (SEQ ID NO: 211), Thielavia terrestris gi|347001418(SEQ ID NO: 212), Magnaporthe oryzae gi|145605493 (SEQ ID NO: 213), M.thermophila gi367032200 (SEQ ID NO: 548), P. chrysogenum gi62816282 (SEQID NO: 549), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a slp5 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 200-213, SEQ ID NOs:548-549. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 200-213, SEQ ID NOs:548-549.

In some embodiments, slp5 is T. reesei slp5. The amino acid sequenceencoded by T. reesei slp5 is set forth in SEQ ID NO: 200. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 200. In further embodiments, the protease has 100% identity toSEQ ID NO: 200.

Slp6

Examples of suitable slp6 proteases include, without limitation,Trichoderma reesei slp6 (SEQ ID NO: 214), T. atroviride jgi|Triat2 (SEQID NO: 215), T. virens jgi|TriviGv29_8_2 (SEQ ID NO: 216), Hypocreavirens gi|29421423 (SEQ ID NO: 217), Hypocrea lixii gi|145583127 (SEQ IDNO: 218), Trichoderma hamatum gi|30144643 (SEQ ID NO: 219), Aspergillusfumigatus gi|2295 (SEQ ID NO: 220), Aspergillus terreus gi|115391147(SEQ ID NO: 221), Aspergillus oryzae gi|169771349 (SEQ ID NO: 222),Aspergillus niger gi|470729 (SEQ ID NO: 223), Glomerella graminicolagi|310794714 (SEQ ID NO: 224), Gibberella zeae gi|46114946 (SEQ ID NO:225), Fusarium oxysporum gi|342873942 (SEQ ID NO: 226), Nectriahaematococca gi|302884541 (SEQ ID NO: 227), Neosartorya fischerigi|119500190 (SEQ ID NO: 228), Verticillium alboatrum gi|302413161 (SEQID NO: 229), Glomerella graminicola gi|310790144 (SEQ ID NO: 230), N.crassa gi85090020 (SEQ ID NO: 550), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a slp6 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 214-230, SEQ ID NO:550. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 214-230, SEQ ID NO:550.

In some embodiments, slp6 is T. reesei slp6. The amino acid sequenceencoded by T. reesei slp6 is set forth in SEQ ID NO: 214. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 214. In further embodiments, the protease has 100% identity toSEQ ID NO: 214.

Slp7

Examples of suitable slp7 proteases include, without limitation,Trichoderma reesei slp7 (SEQ ID NO: 231), T. atroviride jgi|Triat2 (SEQID NO: 232), T. virens jgi|TriviGv29_8_2 (SEQ ID NO: 233), Metarhiziumanisopliae gi|322710320 (SEQ ID NO: 234), Nectria haematococcagi|302915000 (SEQ ID NO: 235), Myceliophthora thermophila gi|347009020,gi367024935 (SEQ ID NO: 236), Gibberella zeae gi|46137655 (SEQ ID NO:237), Thielavia terrestris gi|346996549 (SEQ ID NO: 238), Magnaportheoryzae gi|145610733 (SEQ ID NO: 239), A. nidulans gi67541991 (SEQ ID NO:551), P. chrysogenum gi255933786 (SEQ ID NO: 552), A. niger gi317036543(SEQ ID NO: 553), A. oryzae gi|69782882 (SEQ ID NO: 554), N. crassagi85109979 (SEQ ID NO: 555), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a slp7 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 231-239, SEQ ID NOs:551-555. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 231-239, SEQ ID NOs:551-555.

In some embodiments, slp7 is T. reesei slp7. The amino acid sequenceencoded by T. reesei slp7 is set forth in SEQ ID NO: 231. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 231. In further embodiments, the protease has 100% identity toSEQ ID NO: 231.

Slp8

Examples of suitable slp8 proteases include, without limitation,Trichoderma reesei slp8 (SEQ ID NO: 240), T. atroviridejgi|Triat2|198568 (SEQ ID NO: 241), T. virens jgi|TriviGv29_8_2β3902(SEQ ID NO: 242), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure has an amino acid sequence having 50% or more identity (e.g.60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5% or more to an amino acid sequence selected from SEQ IDNOs: 240-242. In some embodiments, the protease has 100% identity to anamino acid sequence selected from SEQ ID NOs: 240-242.

In some embodiments, slp8 is T. reesei slp8. The amino acid sequenceencoded by T. reesei slp8 is set forth in SEQ ID NO: 240. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 240. In further embodiments, the protease has 100% identity toSEQ ID NO: 240.

Slp Like Proteases

Other slp-like proteases include, without limitation, slp57433 (Tre57433SEQ ID NO:885), slp35726_(Tre35726 SEQ ID NO:886), slp60791_(Tre60791SEQ ID NO:887) or slp109276_(Tre109276 SEQ ID NO:888), and homologsthereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a slp57433, slp35726, slp60791 or slp109276protease, has an amino acid sequence having 50% or more identity (e.g.60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5% or more to an amino acid sequence selected from SEQ IDNOs: 885-888. In some embodiments, the protease has 100% identity to anamino acid sequence selected from SEQ ID NOs: 885-888.

Glutamic Proteases

Glutamic proteases are enzymes that hydrolyze the peptide bonds inpolypeptides and proteins. Glutamic proteases are insensitive topepstatin A, and so are sometimes referred to as pepstatin insensitiveacid proteases. While glutamic proteases were previously grouped withthe aspartic proteases and often jointly referred to as acid proteases,it has been recently found that glutamic proteases have very differentactive site residues than aspartic proteases.

Two glutamic proteases have been identified in Trichoderma fungal cells:gap1 (tre69555) and gap2 (tre106661).

Gap1

Examples of suitable gap1 proteases include, without limitation,Trichoderma reesei gap1 (SEQ ID NO: 118), T. atroviride jgi|Triat2|40863(SEQ ID NO: 119), T. virens jgi|TriviGv29_8_2|192684 (SEQ ID NO: 120),Aspergillus flavus gi|238499183 (SEQ ID NO: 121), Aspergillus nigergi|145251555 (SEQ ID NO: 122), Aspergillus terreus gi|115491521 (SEQ IDNO: 123), gi|37154543 (SEQ ID NO: 124), gi|48425531 (SEQ ID NO: 125),gi|351873 (SEQ ID NO: 126), Thielavia terrestris gi|346997245 (SEQ IDNO: 127), Penicillium chrysogenum gi|255940586 (SEQ ID NO: 128), M.thermophila gi367026504 (SEQ ID NO: 574), A. oryzae gi317150886 (SEQ IDNO: 575), N. crassa gi85097968 (SEQ ID NO: 576), A. niger gi131056 (SEQID NO: 577), P. chrysogenum gi255930123 (SEQ ID NO: 578), A. nigergi145236956 (SEQ ID NO: 579), A. oryzae gi169772955 (SEQ ID NO: 580), A.niger gi145249222 (SEQ ID NO: 581), A. nidulans gi67525839 (SEQ ID NO:582), A. oryzae gi169785367 (SEQ ID NO: 583), P. chrysogenum gi255955319(SEQ ID NO: 584), M. thermophila gi367019352 (SEQ ID NO: 585), A oryzaegi391863974 (SEQ ID NO: 586), M. thermophila gi367024513 (SEQ ID NO:587), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a gap1 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 118-128, SEQ ID NOs:574-587. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 118-128, SEQ ID NOs:574-587.

In some embodiments, gap1 is T. reesei gap1. The amino acid sequenceencoded by T. reesei gap1 is set forth in SEQ ID NO: 118. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 118. In further embodiments, the protease has 100% identity toSEQ ID NO: 118.

Gap2

Examples of suitable gap2 proteases include, without limitation,Trichoderma reesei gap2 (SEQ ID NO: 129), T. atroviridejgi|Triat2|298116 (SEQ ID NO: 130), T. virens jgi|TriviGv29_8_2|30331(SEQ ID NO: 131), jgi|TriviGv29_8_2|225131 (SEQ ID NO: 132), Aspergillusflavus gi|238499183 (SEQ ID NO: 133), Aspergillus niger gi|145251555(SEQ ID NO: 134), Aspergillus nidulans gi|67901056 (SEQ ID NO: 135),Aspergillus clavatus gi|121711990 (SEQ ID NO: 136), Aspergillusfumigatus gi|70986250 (SEQ ID NO: 137), Penicillium marneffeigi|212534108 (SEQ ID NO: 138), Talaromyces stipitatus gi|242789335 (SEQID NO: 139), Grosmannia clavigera gi|320591529 (SEQ ID NO: 140),Neosartorya fischeri gi|119474281 (SEQ ID NO: 141), Penicilliummarneffei gi|212527274 (SEQ ID NO: 142), Penicillium chrysogenumgi|255940586 (SEQ ID NO: 143), gi|131056 (SEQ ID NO: 144), M.thermophila gi367030275 (SEQ ID NO: 588), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a gap2 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 129-144, SEQ ID NO:588. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 129-144, SEQ ID NO:588.

In some embodiments, gap2 is T. reesei gap2. The amino acid sequenceencoded by T. reesei gap2 is set forth in SEQ ID NO: 129. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 129. In further embodiments, the protease has 100% identity toSEQ ID NO: 129.

Other gap-like proteases include, without limitation, gap3 (Tre70927 SEQID NO:889), or gap4_(Tre57575 SEQ ID NO:890), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a gap3 or gap4 protease, has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to anamino acid sequence selected from SEQ ID NO:889 or SEQ ID NO:890respectively. In some embodiments, the protease has 100% identity to anamino acid sequence selected from SEQ ID NOs: 889-889.

Sedolisin Proteases

Sedolisin proteases are enzymes that use a serine residue for hydrolysisof the peptide bonds in polypeptides and proteins. Sedolisin proteasesgenerally contain a unique catalytic triad of serine, glutamate, andaspartate. Sedolisin proteases also contain an aspartate residue in theoxyanion hole. Sedolisin proteases from eukaryotic organisms such asTrichoderma fungi include tripeptidyl peptidase.

Examples of suitable tpp1 proteases include, without limitation,Trichoderma reesei tpp1 (SEQ ID NO: 145), T. atroviridejgi|Triat2|188756 (SEQ ID NO: 146), T. virens jgi|TriviGv29_8_2|217176(SEQ ID NO: 147), Aspergillus fumigatus gi|70993168 (SEQ ID NO: 148),Aspergillus oryzae gi|169776800 (SEQ ID NO: 149), Aspergillus nigergi|145236399 (SEQ ID NO: 150), Aspergillus clavatus gi|121708799 (SEQ IDNO: 151), Aspergillus niger gi|145239871 (SEQ ID NO: 152), Aspergillusclavatus gi|121714541 (SEQ ID NO: 153), Aspergillus terreus gi|115387645(SEQ ID NO: 154), Aspergillus fumigatus gi|70982015 (SEQ ID NO: 155),Sclerotinia sclerotiorum gi|156045898 (SEQ ID NO: 156), Botryotiniafuckeliana gi|154321758 (SEQ ID NO: 157), Neosartorya fischerigi|119499774 (SEQ ID NO: 158), Talaromyces stipitatus gi|242798348 (SEQID NO: 159), Penicillium marneffei gi|212541546 (SEQ ID NO: 160),Gibberella zeae gi|46114460 (SEQ ID NO: 161), Fusarium oxysporumgi|342890694 (SEQ ID NO: 162), Grosmannia clavigera gi|320592937 (SEQ IDNO: 163), Verticillium alboatrum gi|302406186 (SEQ ID NO: 164),Verticillium dahliae gi|346971444 (SEQ ID NO: 165), A. fumigatusCAE51075.1 (SEQ ID NO: 556), A. oryzae XP_001820835.1 (SEQ ID NO: 557),P. chrysogenum XP_002564029.1 (SEQ ID NO: 558), A. nidulans XP_664805.1(SEQ ID NO: 559), P. chrysogenum XP_002565814.1 (SEQ ID NO: 560), M.thermophila XP_003663689.1 (SEQ ID NO: 561), N. crassa XP_958412.1 (SEQID NO: 562), A. niger XP_001394118.1 (SEQ ID NO: 563), A. fumigatusCAE17674.1 (SEQ ID NO: 564), A. niger XP_001400873.1 (SEQ ID NO: 565),A. fumigatus CAE46473.1 (SEQ ID NO: 566), A. oryzae XP_002373530.1 (SEQID NO: 567), A. nidulans XP_660624.1 (SEQ ID NO: 568), P. chrysogenumXP_002562943.1 (SEQ ID NO: 569), A. fumigatus CAE17675.1 (SEQ ID NO:570), A. fumigatus EAL86850.2 (SEQ ID NO: 571), N. crassa XP_961957.1(SEQ ID NO: 572), A. oryzae BAB97387.1 (SEQ ID NO: 573), and homologsthereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a tpp1 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 145-165, SEQ ID NOs:556-573. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 145-165, SEQ ID NOs:556-573.

In some embodiments, tpp1 is T. reesei tpp1. The amino acid sequenceencoded by T. reesei tpp1 is set forth in SEQ ID NO: 145. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 145. In further embodiments, the protease has 100% identity toSEQ ID NO: 145.

Other sedolisin-like proteases include, without limitation, sed2(Tre70962, SEQ ID NO:891), sed3_(Tre81517 SEQ ID NO:892), orsed5_(Tre111838 SEQ ID NO:893), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a sed2, sed3 or sed5 protease, has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to anamino acid sequence selected from SEQ ID NOs: 891-893. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 891-893.

Aminopeptidase Proteases

Aminopeptidases catalyze the cleavage of amino acids from the aminoterminus of protein or peptide substrates. They are widely distributedthroughout the animal and plant kingdoms and are found in manysubcellular organelles, in cytoplasm, and as membrane components. Many,but not all, of these peptidases are zinc metalloenzymes. Amp2 is abifunctional enzyme. It is a leukotriene A4 hydrolase withaminopeptidase activity (EC 3.3.2.6).

Two aminopeptidases have been identified in Trichoderma fungal cells:amp1 (tre81070) and amp2 (tre108592).

Amp1

Examples of suitable amp1 proteases include, without limitation,Trichoderma reesei amp1 81070 (SEQ ID NO: 759), T. virens 74747 (SEQ IDNO: 760), T. atroviride 147450 (SEQ ID NO: 761), F. graminicolaXP_386703.1 (SEQ ID NO: 762), A. nidulans CBF75094.1 (SEQ ID NO: 763),A. niger EHA21022.1 (SEQ ID NO: 764), A. oryzae XP_001727175.1 (SEQ IDNO: 765), A. fumigatus XP_749158.1 (SEQ ID NO: 766), M. thermophilaXP_003667354.1 (SEQ ID NO: 767), F. graminicola XP_385112.1 (SEQ ID NO:768), P. Chrysogenum XP_002567159.1 (SEQ ID NO: 769), A. fumigatusXP_748386.2 (SEQ ID NO: 770), A. oryzae XP_001819545.1 (SEQ ID NO: 771),A. nidulans XP_681714.1 (SEQ ID NO: 772), N. crassa XP_957507.1 (SEQ IDNO: 773), M. thermo XP_003665703.1 (SEQ ID NO: 774), and homologsthereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a amp1 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 759-774. In some embodiments, theprotease has 100% identity to an amino acid sequence selected from SEQID NOs: 759-774.

In some embodiments, amp1 is T. reesei amp1. The amino acid sequenceencoded by T. reesei amp1 is set forth in SEQ ID NO: 759. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 759. In further embodiments, the protease has 100% identity toSEQ ID NO: 759.

Amp2

Examples of suitable amp2 proteases include, without limitation,Trichoderma reesei amp2 108592 (SEQ ID NO: 775), T. virens 73611 (SEQ IDNO: 776), T. atroviride 284076 (SEQ ID NO: 777), F. graminicolaXP_390364.1 (SEQ ID NO: 778), N. crassa XP_960660.1 (SEQ ID NO: 779), M.thermophila XP_003662184.1 (SEQ ID NO: 780), A. oryzae XP_001826499.2(SEQ ID NO: 781), A. niger XP_001390581.1 (SEQ ID NO: 782), A. nidulansXP_663416.1 (SEQ ID NO: 783), A. fumigatus XP_755088.1 (SEQ ID NO: 784),P. chrysogenum XP_002558974.1 (SEQ ID NO: 785) and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a amp2 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 775-785. In some embodiments, theprotease has 100% identity to an amino acid sequence selected from SEQID NOs: 775-785.

In some embodiments, amp2 is T. reesei amp2. The amino acid sequenceencoded by T. reesei amp2 is set forth in SEQ ID NO: 775. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 775. In further embodiments, the protease has 100% identity toSEQ ID NO: 775.

Sep Proteases

Sep proteases are serine proteases belonging to the S28 subtype. Theyhave a catalytic triad of serine, aspartate, and histidine: serine actsas a nucleophile, aspartate as an electrophile, and histidine as a base.These serine proteases include several eukaryotic enzymes such aslysosomal Pro-X carboxypeptidase, dipeptidyl-peptidase II, andthymus-specific serine peptidase.

Examples of suitable sep1 proteases include, without limitation,Trichoderma reesei sep1 124051 (SEQ ID NO: 786), T. virens 39211 (SEQ IDNO: 787), T. atroviride 296922 (SEQ ID NO: 788), A. niger CAK45422.1(SEQ ID NO: 789), A. fumigatus EDP53789.1 (SEQ ID NO: 790), N. crassaXP_958301.1 (SEQ ID NO: 791), M. thermophila XP_003664601.1 (SEQ ID NO:792), M. graminicola XP_384993.1 (SEQ ID NO: 793), M. thermophilaXP_003658945.1 (SEQ ID NO: 794), F. graminicola XP_382380.1 (SEQ ID NO:795), A. niger XP_001395660.1 (SEQ ID NO: 796), M. thermophilaXP_003659734.1 (SEQ ID NO: 797), N. crassa XP_964374.1 (SEQ ID NO: 798),A. fumigatus XP_756068.1 (SEQ ID NO: 799), A. oryzae EIT77098.1 (SEQ IDNO: 800), P. chrysogenum XP_002560028.1 (SEQ ID NO: 801), A. oryzaeEIT71569.1 (SEQ ID NO: 802), A. nidulans CBF79006.1 (SEQ ID NO: 803), A.niger XP_001400740.2 (SEQ ID NO: 804), A. oryzae BAE57999.1 (SEQ ID NO:805), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a sep1 protease, has an amino acid sequence having50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more to an amino acidsequence selected from SEQ ID NOs: 786-805. In some embodiments, theprotease has 100% identity to an amino acid sequence selected from SEQID NOs: 786-805.

In some embodiments, sep1 is T. reesei sep1. The amino acid sequenceencoded by T. reesei sep1 is set forth in SEQ ID NO: 786. In otherembodiments, a protease of the present disclosure has an amino acidsequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more) to SEQID NO: 786. In further embodiments, the protease has 100% identity toSEQ ID NO: 786.

Zinc Metalloprotease

Zinc metalloproteases are protease enzymes that require zinc forcatalytic activity.

Five metalloproteases have been identified in Trichoderma fungal cells:mp1 (tre122703), mp2 (tre122576), mp3 (tre4308), mp4 (tre53343), mp5(tre73809).

mp1, mp2, mp3, mp4 and mp5

Examples of suitable mp1, mp2, mp3, mp4 and mp5 proteases include,without limitation, Trichoderma reesei mp1 (SEQ ID NO: 875), Trichodermareesei mp2 (SEQ ID NO:876), Trichoderma reesei mp3 (SEQ ID NO:877),Trichoderma reesei mp4 (SEQ ID NO:878), Trichoderma reesei mp5 (SEQ IDNO:879), and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure, typically a mp1, mp2, mp3, mp4 or mp5 protease, has an aminoacid sequence having 50% or more identity (e.g. 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or more toan amino acid sequence selected from SEQ ID NOs:875-879. In someembodiments, the protease has 100% identity to an amino acid sequenceselected from SEQ ID NOs: 875-879.

Other Proteases

Examples of other suitable proteases include, without limitation,

-   -   Trichoderma reesei Group A protease selected from the group of        protease65735 (SEQ ID NO:894), protease77577 (SEQ ID NO:895),        protease81087 (SEQ ID NO:896), protease56920 (SEQ ID NO:900),        protease122083 (SEQ ID NO:911), protease79485 (SEQ ID NO:910),        protease120998 (SEQ ID NO:901), or protease61127 (SEQ ID NO:912;    -   Trichoderma reesei Group B protease selected from the group of        protease21659 (SEQ ID NO:905), protease58387 (SEQ ID NO:921),        protease75159 (SEQ ID NO:918), protease56853 (SEQ ID NO:914), or        protease64193 (SEQ ID NO:908);    -   Trichoderma reesei Group C protease selected from the group of        protease82452 (SEQ ID NO:906), protease80762 (SEQ ID NO:913),        protease21668 (SEQ ID NO:919), protease81115 (SEQ ID NO:907),        protease82141 (SEQ ID NO:902), protease23475 (SEQ ID NO:909);    -   Trichoderma reesei Group D protease selected from the group of        protease121890 (903), protease22718 (SEQ ID NO:904),        protease47127 (SEQ ID NO:899), protease61912 (SEQ ID NO:920),        protease80843 (SEQ ID NO:897), protease66608 (SEQ ID NO:923),        protease72612 (SEQ ID NO:898), protease40199 (SEQ ID NO:917); or    -   Trichoderma reesei Group E protease selected from the group of        protease22210 (SEQ ID NO:915), protease111694 (SEQ ID NO:916),        protease82577 (SEQ ID NO:922),        and homologs thereof.

Accordingly, in certain embodiments, a protease of the presentdisclosure has an amino acid sequence having 50% or more identity (e.g.60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, 99.5% or more to an amino acid sequence selected from SEQ IDNOs: 894-923). In some embodiments, the protease has 100% identity to anamino acid sequence selected from SEQ ID NOs: 894-923.

Homologous Proteases

Other embodiments of the present disclosure relate to reducing theactivity of proteases that are homologous to the proteases of thepresent disclosure. “Homology” as used herein refers to sequencesimilarity between a reference sequence and at least a fragment of asecond sequence. Homologs may be identified by any method known in theart, preferably, by using the BLAST tool to compare a reference sequenceto a single second sequence or fragment of a sequence or to a databaseof sequences. As described below, BLAST will compare sequences basedupon percent identity and similarity.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acid or amino acid sequences, refers to two or moresequences or subsequences that are the same. Two sequences are“substantially identical” if two sequences have a specified percentageof amino acid residues or nucleotides that are the same (i.e., 29%identity, optionally 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, 99% or 100% identity over a specified region, or, whennot specified, over the entire sequence), when compared and aligned formaximum correspondence over a comparison window, or designated region asmeasured using one of the following sequence comparison algorithms or bymanual alignment and visual inspection. Optionally, the identity existsover a region that is at least about 50 nucleotides (or 10 amino acids)in length, or more preferably over a region that is 100 to 500 or 1000or more nucleotides (or 20, 50, 200, or more amino acids) in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters. When comparing two sequences foridentity, it is not necessary that the sequences be contiguous, but anygap would carry with it a penalty that would reduce the overall percentidentity. For blastn, the default parameters are Gap opening penalty=5and Gap extension penalty=2. For blastp, the default parameters are Gapopening penalty=11 and Gap extension penalty=1.

A “comparison window,” as used herein, includes reference to a segmentof any one of the number of contiguous positions including, but notlimited to from 20 to 600, usually about 50 to about 200, more usuallyabout 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith and Waterman (1981), by the homology alignment algorithm ofNeedleman and Wunsch (1970) J Mol Biol 48(3):443-453, by the search forsimilarity method of Pearson and Lipman (1988) Proc Natl Acad Sci USA85(8):2444-2448, by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or bymanual alignment and visual inspection [see, e.g., Brent et al., (2003)Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (RingbouEd)].

Two examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1997) Nucleic AcidsRes 25(17):3389-3402 and Altschul et al. (1990) J. Mol Biol215(3)-403-410, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) or 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix [see Henikoff and Henikoff, (1992) Proc Natl Acad Sci USA89(22):10915-109191 alignments (B) of 50, expectation (E) of 10, M=5,N=−4, and a comparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul, (1993)Proc Natl Acad Sci USA 90(12):5873-5877). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

Other than percentage of sequence identity noted above, anotherindication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross-reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, for example, where the two peptidesdiffer only by conservative substitutions. Another indication that twonucleic acid sequences are substantially identical is that the twomolecules or their complements hybridize to each other under stringentconditions, as described below. Yet another indication that two nucleicacid sequences are substantially identical is that the same primers canbe used to amplify the sequence.

As disclosed herein, proteases of the present disclosure may alsoinclude proteases that are conservatively modified variants of proteasesencoded by the protease genes disclosed above. “Conservatively modifiedvariants” as used herein include individual substitutions, deletions oradditions to an encoded amino acid sequence which result in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the disclosure. The following eight groupscontain amino acids that are conservative substitutions for oneanother: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamicacid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6)Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S),Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g.,Creighton, Proteins (1984)).

Phylogenetic trees of aspartic, subtilisin, glutamic and sedolisinproteases of selected filamentous fungi are described in WO2013/102674.

Methods of Reducing the Activity of Proteases of the Invention

Further aspects of the present disclosure relate to reducing theactivity of proteases found in filamentous fungal cells that express aheterologous polypeptide, such a mammalian polypeptide.

The activity of proteases found in filamentous fungal cells can bereduced by any method known to those of skill in the art.

In some embodiments reduced activity of proteases is achieved byreducing the expression of the protease, for example, by promotermodification or RNAi.

In other embodiments, reduced activity of proteases is achieved bymodifying the gene encoding the protease. Examples of such modificationsinclude, without limitation, a knock-out mutation, a truncationmutation, a point mutation, a missense mutation, a substitutionmutation, a frameshift mutation, an insertion mutation, a duplicationmutation, an amplification mutation, a translocation mutation, or aninversion mutation, and that results in a reduction in the correspondingprotease activity. Methods of generating at least one mutation in aprotease encoding gene of interest are well known in the art andinclude, without limitation, random mutagenesis and screening,site-directed mutagenesis, PCR mutagenesis, insertional mutagenesis,chemical mutagenesis, and irradiation.

In certain embodiments, a portion of the protease encoding gene ismodified, such as the region encoding the catalytic domain, the codingregion, or a control sequence required for expression of the codingregion. Such a control sequence of the gene may be a promoter sequenceor a functional part thereof, i.e., a part that is sufficient foraffecting expression of the gene. For example, a promoter sequence maybe inactivated resulting in no expression or a weaker promoter may besubstituted for the native promoter sequence to reduce expression of thecoding sequence. Other control sequences for possible modificationinclude, without limitation, a leader sequence, a propeptide sequence, asignal sequence, a transcription terminator, and a transcriptionalactivator.

Protease encoding genes of the present disclosure that are present infilamentous fungal cells that express a recombinant polypeptide may alsobe modified by utilizing gene deletion techniques to eliminate or reduceexpression of the gene. Gene deletion techniques enable the partial orcomplete removal of the gene thereby eliminating their expression. Insuch methods, deletion of the gene may be accomplished by homologousrecombination using a plasmid that has been constructed to contiguouslycontain the 5′ and 3′ regions flanking the gene.

The protease encoding genes of the present disclosure that are presentin filamentous fungal cells that express a recombinant polypeptide mayalso be modified by introducing, substituting, and/or removing one ormore nucleotides in the gene, or a control sequence thereof required forthe transcription or translation of the gene. For example, nucleotidesmay be inserted or removed for the introduction of a stop codon, theremoval of the start codon, or a frame-shift of the open reading frame.Such a modification may be accomplished by methods known in the art,including without limitation, site-directed mutagenesis and peRgenerated mutagenesis (see, for example, Botstein and Shortie, 1985,Science 229: 4719; Lo et al., 1985, Proceedings of the National Academyof Sciences USA 81: 2285; Higuchi et al., 1988, Nucleic Acids Research16: 7351; Shimada, 1996, Meth. Mol. Biol. 57: 157; Ho et al., 1989, Gene77: 61; Horton et al., 1989, Gene 77: 61; and Sarkar and Sommer, 1990,BioTechniques 8: 404).

Additionally, protease encoding genes of the present disclosure that arepresent in filamentous fungal cells that express a recombinantpolypeptide may be modified by gene disruption techniques by insertinginto the gene a disruptive nucleic acid construct containing a nucleicacid fragment homologous to the gene that will create a duplication ofthe region of homology and incorporate construct DNA between theduplicated regions. Such a gene disruption can eliminate gene expressionif the inserted construct separates the promoter of the gene from thecoding region or interrupts the coding sequence such that anonfunctional gene product results. A disrupting construct may be simplya selectable marker gene accompanied by 5′ and 3′ regions homologous tothe gene. The selectable marker enables identification of transformantscontaining the disrupted gene.

Protease encoding genes of the present disclosure that are present infilamentous fungal cells that express a recombinant polypeptide may alsobe modified by the process of gene conversion (see, for example,Iglesias and Trautner, 1983, Molecular General Genetics 189:5 73-76).For example, in the gene conversion a nucleotide sequence correspondingto the gene is mutagenized in vitro to produce a defective nucleotidesequence, which is then transformed into a Trichoderma strain to producea defective gene. By homologous recombination, the defective nucleotidesequence replaces the endogenous gene. It may be desirable that thedefective nucleotide sequence also contains a marker for selection oftransformants containing the defective gene.

Protease encoding genes of the present disclosure that are present infilamentous fungal cells that express a recombinant polypeptide may alsobe modified by established anti-sense techniques using a nucleotidesequence complementary to the nucleotide sequence of the gene (see, forexample, Parish and Stoker, 1997, FEMS Microbiology Letters 154:151-157). In particular, expression of the gene by filamentous fungalcells may be reduced or inactivated by introducing a nucleotide sequencecomplementary to the nucleotide sequence of the gene, which may betranscribed in the strain and is capable of hybridizing to the mRNAproduced in the cells. Under conditions allowing the complementaryanti-sense nucleotide sequence to hybridize to the mRNA, the amount ofprotein translated is thus reduced or eliminated.

In addition, protease encoding genes of the present disclosure that arepresent in filamentous fungal cells that express a recombinantpolypeptide may also be modified by established RNA interference (RNAi)techniques (see, for example, WO 2005/056772 and WO 2008/080017).

Protease encoding genes of the present disclosure that are present infilamentous fungal cells that express a recombinant polypeptide may alsobe modified by random or specific mutagenesis using methods well knownin the art, including without limitation, chemical mutagenesis (see, forexample, Hopwood, The Isolation of Mutants in Methods in Microbiology(J. R. Norris and D. W. Ribbons, eds.) pp. 363-433, Academic Press, NewYork, 25 1970). Modification of the gene may be performed by subjectingfilamentous fungal cells to mutagenesis and screening for mutant cellsin which expression of the gene has been reduced or inactivated. Themutagenesis, which may be specific or random, may be performed, forexample, by use of a suitable physical or chemical mutagenizing agent,use of a suitable oligonucleotide, subjecting the DNA sequence to peRgenerated mutagenesis, or any combination thereof. Examples of physicaland chemical mutagenizing agents include, without limitation,ultraviolet (UV) irradiation, hydroxylamine,N-methyl-N′-nitro-N-nitrosoguanidine (MNNG),N-methyl-N′-nitrosogaunidine (NTG) O-methyl hydroxylamine, nitrous acid,ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, andnucleotide analogues. When such agents are used, the mutagenesis istypically performed by incubating the Trichoderma cells to bemutagenized in the presence of the mutagenizing agent of choice undersuitable conditions, and then selecting for mutants exhibiting reducedor no expression of the gene.

In certain embodiments, the at least one mutation or modification in aprotease encoding gene of the present disclosure results in a modifiedprotease that has no detectable protease activity. In other embodiments,the at least one modification in a protease encoding gene of the presentdisclosure results in a modified protease that has at least 25% less, atleast 50% less, at least 75% less, at least 90%, at least 95%, at least100%, at least 200%, at least 300%, at least 400%, at least 500%, atleast 600%, at least 700%, at least 800%, at least 900%, at least1,000%, or a higher percentage less protease activity compared to acorresponding non-modified protease.

In certain embodiments, for example, in a Trichoderma cell, the at leastone mutation or modification in a protease encoding gene of the presentdisclosure results in a reduction of total protease activity to 49% orless, typically with a mutation in at least 2 distinct protease genes,or 31% or less, typically with a mutation in at least 3 distinctprotease genes, or 13% or less, typically with a mutation in at least 4distinct protease genes, or 10% or less, typically with a mutation in atleast 5 distinct protease genes, or 6.3% or less, typically with amutation in at least 6 distinct protease genes, or 5.5% or less,typically with a mutation in at least 7 distinct protease genes, of thetotal protease activity of the corresponding parental Trichoderma cell.

Heterologous Polypeptides of the Invention

The invention herein further relates to increasing the production ofheterologous polypeptides in filamentous fungal cells that express suchheterologous polypeptides by reducing the activity of proteases found inthe cells.

As used herein a “heterologous polypeptide” refers to a polypeptide thatis not naturally found in (i.e., endogenous) a filamentous fungal cellof the present disclosure, or that is expressed at an elevated level ina filamentous fungal cell as compared to the endogenous version of thepolypeptide. In certain embodiments, the heterologous polypeptide is amammalian polypeptide. In other embodiments, the heterologouspolypeptide is a non-mammalian polypeptide.

Mammalian Polypeptides

Mammalian polypeptides of the present disclosure may be any mammalianpolypeptide having a biological activity of interest. As used herein, a“mammalian polypeptide” is a polypeptide that is natively expressed in amammal, a polypeptide that is derived from a polypeptide that isnatively expressed in a mammal, or a fragment thereof. A mammalianpolypeptide also includes peptides and oligopeptides that retainbiological activity. Mammalian polypeptides of the present disclosuremay also include two or more polypeptides that are combined to form theencoded product. Mammalian polypeptides of the present disclosure mayfurther include fusion polypeptides, which contain a combination ofpartial or complete amino acid sequences obtained from at least twodifferent polypeptides. Mammalian polypeptides may also includenaturally occurring allelic and engineered variations of any of thedisclosed mammalian polypeptides and hybrid mammalian polypeptides.

The mammalian polypeptide may be a naturally glycosylated polypeptide ora naturally non-glycosylated polypeptide.

Examples of suitable mammalian polypeptides include, without limitation,immunoglobulins, antibodies, antigens, antimicrobial peptides, enzymes,growth factors, hormones, interferons, cytokines, interleukins,immunodilators, neurotransmitters, receptors, reporter proteins,structural proteins, and transcription factors.

Specific examples of suitable mammalian polypeptides include, withoutlimitation, immunoglobulins, immunoglobulin heavy chains, immunoglobulinlight chains, monoclonal antibodies, hybrid antibodies, F(ab′)2 antibodyfragments, F(ab) antibody fragments, Fv molecules, single-chain Fvantibodies, dimeric antibody fragments, trimeric antibody fragments,functional antibody fragments, immunoadhesins, insulin-like growthfactor 1, growth hormone, insulin, interferon alpha 2b, fibroblastgrowth factor 21, human serum albumin, camelid antibodies and/orantibody fragments, single domain antibodies, multimeric single domainantibodies, and erythropoietin.

Other examples of suitable mammalian proteins include, withoutlimitation, an oxidoreductase, a transferase, a hydrolase, a lyase, anisomerase, a ligase, an aminopeptidase, an amylase, a carbohydrase, acarboxypeptidase, a catalase, a glycosyltransferase, adeoxyribonuclease, an esterase, a galactosidase, a betagalactosidase, aglucosidase, a glucuronidase, a glucuronoyl esterase, a haloperoxidase,an invertase, a lipase, an oxidase, a phospholipase, a proteolyticenzyme, a ribonuclease, a urokinase, an albumin, a collagen, atropoelastin, and an elastin.

Non-Mammalian Polypeptides

Non-mammalian polypeptides of the present disclosure may be anynon-mammalian polypeptide having a biological activity of interest. Asused herein, a “non-mammalian polypeptide” is a polypeptide that isnatively expressed in a non-mammalian organism, such as a fungal cell, apolypeptide that is derived from a polypeptide that is nativelyexpressed in a non-mammal organism, or a fragment thereof. Anon-mammalian polypeptide also includes peptides and oligopeptides thatretain biological activity. Non-mammalian polypeptides of the presentdisclosure may also include two or more polypeptides that are combinedto form the encoded product. Non-mammalian polypeptides of the presentdisclosure may further include fusion polypeptides, which contain acombination of partial or complete amino acid sequences obtained from atleast two different polypeptides. Non-mammalian polypeptides may alsoinclude naturally occurring allelic and engineered variations of any ofthe disclosed non-mammalian polypeptides and hybrid non-mammalianpolypeptides.

Examples of suitable non-mammalian polypeptides include, withoutlimitation, aminopeptidases, amylases, carbohydrases, carboxypeptidases,catalases, cellulases, chitinases, cutinases, deoxyribonucleases,esterases, alpha-galactosidases, beta-galactosidases, glucoamylases,alpha-glucosidases, beta-glucosidases, invertases, laccases, lipases,mutanases, oxidases, pectinolytic enzymes, peroxidases, phospholipases,phytases, polyphenoloxidases, proteolytic enzymes, ribonucleases,transglutaminases and xylanases.

Heterologous Polypeptide Production

A heterologous polypeptide of interest is produced by filamentous fungalcells of the present disclosure containing at least three proteaseshaving reduced activity by cultivating the cells in a nutrient mediumfor production of the heterologous polypeptide using methods known inthe art. For example, the cells may be cultivated by shake flaskcultivation, small-scale or large-scale fermentation (includingcontinuous, batch, fed-batch, or solid state fermentations) inlaboratory or industrial fermentors performed in a suitable medium andunder conditions allowing the polypeptide to be expressed and/orisolated. The cultivation takes place in a suitable nutrient mediumcomprising carbon and nitrogen sources and inorganic salts, usingprocedures known in the art. Suitable media are available fromcommercial suppliers or may be prepared according to publishedcompositions (e.g., in catalogues of the American Type CultureCollection). The secreted polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it may be obtained from celllysates.

A heterologous polypeptide of interest produced by a filamentous fungalcell of the present disclosure containing at least three proteaseshaving reduced activity may be detected using methods known in the artthat are specific for the heterologous polypeptide. These detectionmethods may include, without limitation, use of specific antibodies,high performance liquid chromatography, capillary chromatography,formation of an enzyme product, disappearance of an enzyme substrate,and SDS-PAGE. For example, an enzyme assay may be used to determine theactivity of an enzyme. Procedures for determining enzyme activity areknown in the art for many enzymes (see, for example, O. Schomburg and M.Salzmann (eds.), Enzyme Handbook, Springer-Verlag, New York, 1990).

The resulting heterologous polypeptide may be isolated by methods knownin the art. For example, a heterologous polypeptide of interest may beisolated from the cultivation medium by conventional proceduresincluding, without limitation, centrifugation, filtration, extraction,spray-drying, evaporation, and precipitation. The isolated heterologouspolypeptide may then be further purified by a variety of proceduresknown in the art including, without limitation, chromatography (e.g.,ion exchange, affinity, hydrophobic, chromatofocusing, and sizeexclusion), electrophoretic procedures (e.g., preparative isoelectricfocusing (IEF), differential solubility (e g, ammonium sulfateprecipitation), or extraction (see, for example, Protein Purification,J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989).

Preparation of Polynucleotides Encoding Heterologous Polypeptides

Sequences of the heterologous polynucleotides of the present disclosureare prepared by any suitable method known in the art, including, withoutlimitation, direct chemical synthesis or cloning. For direct chemicalsynthesis, formation of a polymer of nucleic acids typically involvessequential addition of 3 ‘-blocked and 5’-blocked nucleotide monomers tothe terminal 5′-hydroxyl group of a growing nucleotide chain, whereineach addition is effected by nucleophilic attack of the terminal5′-hydroxyl group of the growing chain on the 3′-position of the addedmonomer, which is typically a phosphorus derivative, such as aphosphotriester, phosphoramidite, or the like. Such methodology is knownto those of ordinary skill in the art and is described in the pertinenttexts and literature [e.g., in Matteucci et al., (1980) Tetrahedron Lett21:719-722; U.S. Pat. Nos. 4,500,707; 5,436,327; and 5,700,637]. Inaddition, the desired sequences may be isolated from natural sources bysplitting DNA using appropriate restriction enzymes, separating thefragments using gel electrophoresis, and thereafter, recovering thedesired nucleic acid sequence from the gel via techniques known to thoseof ordinary skill in the art, such as utilization of polymerase chainreactions (PCR; e.g., U.S. Pat. No. 4,683,195).

Each heterologous polynucleotide of the present disclosure can beincorporated into an expression vector. “Expression vector” or “vector”refers to a compound and/or composition that transduces, transforms, orinfects a host cell, thereby causing the cell to express nucleic acidsand/or proteins other than those native to the cell, or in a manner notnative to the cell. An “expression vector” contains a sequence ofnucleic acids (ordinarily RNA or DNA) to be expressed by the host cell.Optionally, the expression vector also includes materials to aid inachieving entry of the nucleic acid into the host cell, such as a virus,liposome, protein coating, or the like. The expression vectorscontemplated for use in the present disclosure include those into whicha nucleic acid sequence can be inserted, along with any preferred orrequired operational elements. Further, the expression vector must beone that can be transferred into a host cell and replicated therein.Preferred expression vectors are plasmids, particularly those withrestriction sites that have been well documented and that contain theoperational elements preferred or required for transcription of thenucleic acid sequence. Such plasmids, as well as other expressionvectors, are well known in the art.

Incorporation of the individual polynucleotides may be accomplishedthrough known methods that include, for example, the use of restrictionenzymes (such as BamHI, EcoRI, HhaI, XhoI, XmaI, and so forth) to cleavespecific sites in the expression vector, e.g., plasmid. The restrictionenzyme produces single stranded ends that may be annealed to apolynucleotide having, or synthesized to have, a terminus with asequence complementary to the ends of the cleaved expression vector.Annealing is performed using an appropriate enzyme, e.g., DNA ligase. Aswill be appreciated by those of ordinary skill in the art, both theexpression vector and the desired polynucleotide are often cleaved withthe same restriction enzyme, thereby assuring that the ends of theexpression vector and the ends of the polynucleotide are complementaryto each other. In addition, DNA linkers maybe used to facilitate linkingof nucleic acids sequences into an expression vector.

A series of individual polynucleotides can also be combined by utilizingmethods that are known t in the art (e.g., U.S. Pat. No. 4,683,195).

For example, each of the desired polynucleotides can be initiallygenerated in a separate PCR. Thereafter, specific primers are designedsuch that the ends of the PCR products contain complementary sequences.When the PCR products are mixed, denatured, and reannealed, the strandshaving the matching sequences at their 3′ ends overlap and can act asprimers for each other. Extension of this overlap by DNA polymeraseproduces a molecule in which the original sequences are “spliced”together. In this way, a series of individual polynucleotides may be“spliced” together and subsequently transduced into a host cellsimultaneously. Thus, expression of each of the plurality ofpolynucleotides is affected.

Individual polynucleotides, or “spliced” polynucleotides, are thenincorporated into an expression vector. The present disclosure is notlimited with respect to the process by which the polynucleotide isincorporated into the expression vector. Those of ordinary skill in theart are familiar with the necessary steps for incorporating apolynucleotide into an expression vector. A typical expression vectorcontains the desired polynucleotide preceded by one or more regulatoryregions, along with a ribosome binding site, e.g., a nucleotide sequencethat is 3-9 nucleotides in length and located 3-11 nucleotides upstreamof the initiation codon in E. coli. See Shine and Dalgarno (1975) Nature254(5495):34-38 and Steitz (1979) Biological Regulation and Development(ed. Goldberger, R. F.), 1:349-399 (Plenum, New York).

The term “operably linked” as used herein refers to a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of the DNA sequence or polynucleotide such thatthe control sequence directs the expression of a polypeptide.

Regulatory regions include, for example, those regions that contain apromoter and an operator. A promoter is operably linked to the desiredpolynucleotide, thereby initiating transcription of the polynucleotidevia an RNA polymerase enzyme. An operator is a sequence of nucleic acidsadjacent to the promoter, which contains a protein-binding domain wherea repressor protein can bind. In the absence of a repressor protein,transcription initiates through the promoter. When present, therepressor protein specific to the protein-binding domain of the operatorbinds to the operator, thereby inhibiting transcription. In this way,control of transcription is accomplished, based upon the particularregulatory regions used and the presence or absence of the correspondingrepressor protein. Examples include lactose promoters (Lad repressorprotein changes conformation when contacted with lactose, therebypreventing the Lad repressor protein from binding to the operator) andtryptophan promoters (when complexed with tryptophan, TrpR repressorprotein has a conformation that binds the operator; in the absence oftryptophan, the TrpR repressor protein has a conformation that does notbind to the operator). Another example is the tac promoter (see de Boeret al., (1983) Proc Natl Acad Sci USA 80(1):21-25). As will beappreciated by those of ordinary skill in the art, these and otherexpression vectors may be used in the present disclosure, and thepresent disclosure is not limited in this respect.

Although any suitable expression vector may be used to incorporate thedesired sequences, readily available expression vectors include, withoutlimitation: plasmids, such as pSClOl, pBR322, pBBR1MCS-3, pUR, pEX,pMR100, pCR4, pBAD24, pUC19, pRS426; and bacteriophages, such as M13phage and λ phage. Of course, such expression vectors may only besuitable for particular host cells. One of ordinary skill in the art,however, can readily determine through routine experimentation whetherany particular expression vector is suited for any given host cell. Forexample, the expression vector can be introduced into the host cell,which is then monitored for viability and expression of the sequencescontained in the vector. In addition, reference may be made to therelevant texts and literature, which describe expression vectors andtheir suitability to any particular host cell.

Suitable expression vectors for the purposes of the invention, includingthe expression of the desired heterologous polypeptide, enzyme, and oneor more catalytic domains described herein, include expression vectorscontaining the polynucleotide encoding the desired heterologouspolypeptide, enzyme, or catalytic domain(s) operably linked to aconstitutive or an inducible promoter. Examples of particularly suitablepromoters for operable linkage to such polynucleotides include promotersfrom the following genes: gpdA, cbh1, Aspergillus oryzae TAKA amylase,Rhizomucor miehei aspartic proteinase, Aspergillus niger neutralalpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillusniger glucoamylase (glaA), Aspergillus awamori glaA, Rhizomucor mieheilipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, Aspergillusoryzae acetamidase, Fusarium oxysporum trypsin-like protease, fungalendo α-L-arabinase (abnA), fungal α-L-arabinofuranosidase A (abfA),fungal α-L-arabinofuranosidase B (abfB), fungal xylanase (xlnA), fungalphytase, fungal ATP-synthetase, fungal subunit 9 (oliC), fungal triosephosphate isomerase (tpi), fungal alcohol dehydrogenase (adhA), fungalα-amylase (amy), fungal amyloglucosidase (glaA), fungal acetamidase(amdS), fungal glyceraldehyde-3-phosphate dehydrogenase (gpd), yeastalcohol dehydrogenase, yeast lactase, yeast 3-phosphoglycerate kinase,yeast triosephosphate isomerase, bacterial α-amylase, bacterial Spo2,and SSO. Examples of such suitable expression vectors and promoters arealso described in WO2012/069593, the entire contents of which is herebyincorporated by reference herein.

Pharmaceutical Compositions Containing Heterologous PolypeptidesProduced by Filamentous Fungal Cells of the Invention

In another aspect, the present invention provides a composition, e.g., apharmaceutical composition, containing one or more heterologouspolypeptides of interest, such as mammalian polypeptides, produced bythe filamentous fungal cells of the present disclosure having reducedactivity of at least three proteases and further containing arecombinant polynucleotide encoding the heterologous polypeptide,formulated together with a pharmaceutically acceptable carrier.Pharmaceutical compositions of the invention also can be administered incombination therapy, i.e., combined with other agents. For example, thecombination therapy can include a mammalian polypeptide of interestcombined with at least one other therapeutic agent.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., the mammalianpolypeptide of interest, may be coated in a material to protect thecompound from the action of acids and other natural conditions that mayinactivate the compound.

The pharmaceutical compositions of the invention may include one or morepharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to a salt that retains the desired biological activity of theparent compound and does not impart any undesired toxicological effects(see e.g., Berge, S. M., et al. (1977) J. Pharm. Sci. 66:1-19). Examplesof such salts include acid addition salts and base addition salts. Acidaddition salts include those derived from nontoxic inorganic acids, suchas hydrochloric, nitric, phosphoric, sulfuric, hydrobromic, hydroiodic,phosphorous and the like, as well as from nontoxic organic acids such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromaticsulfonic acids and the like. Base addition salts include those derivedfrom alkaline earth metals, such as sodium, potassium, magnesium,calcium and the like, as well as from nontoxic organic amines, such asN,N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine,choline, diethanolamine, ethylenediamine, procaine and the like.

A pharmaceutical composition of the invention also may also include apharmaceutically acceptable antioxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, and by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, phenol sorbic acid, and thelike. It may also be desirable to include isotonic agents, such assugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe invention is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the certain methods of preparation arevacuum drying and freeze-drying (lyophilization) that yield a powder ofthe active ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

For administration of a mammalian polypeptide of interest, in particularwhere the mammalian polypeptide is an antibody, the dosage ranges fromabout 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the hostbody weight. For example, dosages can be 0.3 mg/kg body weight, 1 mg/kgbody weight, 3 mg/kg body weight, 5 mg/kg body weight or 10 mg/kg bodyweight or within the range of 1-10 mg/kg. An exemplary treatment regimeentails administration once per week, once every two weeks, once everythree weeks, once every four weeks, once a month, once every 3 months oronce every three to 6 months. Certain dosage regimens for an antibodymay include 1 mg/kg body weight or 3 mg/kg body weight via intravenousadministration, with the antibody being given using one of the followingdosing schedules: (i) every four weeks for six dosages, then every threemonths; (ii) every three weeks; (iii) 3 mg/kg body weight once followedby 1 mg/kg body weight every three weeks.

Alternatively a mammalian polypeptide of interest can be administered asa sustained release formulation, in which case less frequentadministration is required. Dosage and frequency vary depending on thehalf-life of the administered substance in the patient. In general,human antibodies show the longest half life, followed by humanizedantibodies, chimeric antibodies, and nonhuman antibodies. The dosage andfrequency of administration can vary depending on whether the treatmentis prophylactic or therapeutic. In prophylactic applications, arelatively low dosage is administered at relatively infrequent intervalsover a long period of time. Some patients continue to receive treatmentfor the rest of their lives. In therapeutic applications, a relativelyhigh dosage at relatively short intervals is sometimes required untilprogression of the disease is reduced or terminated, and preferablyuntil the patient shows partial or complete amelioration of symptoms ofdisease. Thereafter, the patient can be administered a prophylacticregime.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of the present disclosure may be varied so as to obtain anamount of the active ingredient which is effective to achieve thedesired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient. The selecteddosage level will depend upon a variety of pharmacokinetic factorsincluding the activity of the particular compositions of the presentinvention employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular compositions employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A “therapeutically effective dosage” of an immunoglobulin of the presentdisclosure preferably results in a decrease in severity of diseasesymptoms, an increase in frequency and duration of disease symptom-freeperiods, or a prevention of impairment or disability due to the diseaseaffliction. For example, for the treatment of tumors, a “therapeuticallyeffective dosage” preferably inhibits cell growth or tumor growth by atleast about 20%, more preferably by at least about 40%, even morepreferably by at least about 60%, and still more preferably by at leastabout 80% relative to untreated subjects. The ability of a compound toinhibit tumor growth can be evaluated in an animal model systempredictive of efficacy in human tumors. Alternatively, this property ofa composition can be evaluated by examining the ability of the compoundto inhibit, such inhibition in vitro by assays known to the skilledpractitioner. A therapeutically effective amount of a therapeuticcompound can decrease tumor size, or otherwise ameliorate symptoms in asubject. One of ordinary skill in the art would be able to determinesuch amounts based on such factors as the subject's size, the severityof the subject's symptoms, and the particular composition or route ofadministration selected.

A composition of the present disclosure can be administered via one ormore routes of administration using one or more of a variety of methodsknown in the art. As will be appreciated by the skilled artisan, theroute and/or mode of administration will vary depending upon the desiredresults. Certain routes of administration for binding moieties of theinvention include intravenous, intramuscular, intradermal,intraperitoneal, subcutaneous, spinal or other parenteral routes ofadministration, for example by injection or infusion. The phrase“parenteral administration” as used herein means modes of administrationother than enteral and topical administration, usually by injection, andincludes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion.

Alternatively, a mammalian polypeptide according to the presentdisclosure can be administered via a nonparenteral route, such as atopical, epidermal or mucosal route of administration, for example,intranasally, orally, vaginally, rectally, sublingually or topically.

The active compounds can be prepared with carriers that will protect thecompound against rapid release, such as a controlled releaseformulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. (see, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978).

Therapeutic compositions can be administered with medical devices knownin the art. For example, in a certain embodiment, a therapeuticcomposition of the invention can be administered with a needlelesshypodermic injection device, such as the devices disclosed in U.S. Pat.Nos. 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824;or 4,596,556. Examples of well-known implants and modules useful in thepresent invention include: U.S. Pat. No. 4,487,603, which discloses animplantable micro-infusion pump for dispensing medication at acontrolled rate; U.S. Pat. No. 4,486,194, which discloses a therapeuticdevice for administering medicants through the skin; U.S. Pat. No.4,447,233, which discloses a medication infusion pump for deliveringmedication at a precise infusion rate; U.S. Pat. No. 4,447,224, whichdiscloses a variable flow implantable infusion apparatus for continuousdrug delivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system.

In certain embodiments, the use of mammalian polypeptides according tothe present disclosure is for the treatment of any disease that may betreated with therapeutic antibodies.

Filamentous Fungal Cells of the Invention

The invention herein also relates to increasing the levels of productionof heterologous polypeptides, such as mammalian polypeptides, infilamentous fungal cells by reducing or eliminating the activity of atleast three proteases found in cells that express heterologouspolypeptides, and that catalyze the degradation of the heterologouspolypeptides. Reducing or eliminating the activity of proteases found inthe filamentous fungal cells that express heterologous polypeptidesincreases the stability of the expressed recombinant polypeptides, whichresults in an increased level of production of the heterologouspolypeptides. The activity of the proteases found in the filamentousfungal cells may be reduced, for example, by modifying the genesencoding the proteases.

“Filamentous fungal cells” include cells from all filamentous forms ofthe subdivision Eumycota and Oomycota (as defined by Hawksworth et al.,In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995,CAB International, University Press, Cambridge, UK). Filamentous fungalcells are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

Any filamentous fungal cell may be used in the present disclosure solong as it remains viable after being transformed with a sequence ofnucleic acids and/or being modified or mutated to decrease proteaseactivity. Preferably, the filamentous fungal cell is not adverselyaffected by the transduction of the necessary nucleic acid sequences,the subsequent expression of the proteins (e.g., mammalian proteins), orthe resulting intermediates.

Examples of suitable filamentous fungal cells include, withoutlimitation, cells from an Acremonium, Aspergillus, Fusarium, Humicola,Mucor, Myceliophthora, Neurospora, Penicillium, Scytalidium, Thielavia,Tolypocladium, or Trichoderma strain. In certain embodiments, thefilamentous fungal cell is from a Trichoderma sp., Acremonium,Aspergillus, Aureobasidium, Cryptococcus, Chrysosporium, Chrysosporiumlucknowense, Filibasidium, Fusarium, Gibberella, Magnaporthe, Mucor,Myceliophthora, Myrothecium, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus,Thielavia, or Tolypocladium strain.

Aspergillus fungal cells of the present disclosure may include, withoutlimitation, Aspergillus aculeatus, Aspergillus awamori, Aspergillusclavatus, Aspergillus flavus, Aspergillus foetidus, Aspergillusfumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillusniger, Aspergillus oryzae, or Aspergillus terreus.

Neurospora fungal cells of the present disclosure may include, withoutlimitation, Neurospora crassa.

In certain embodiments, the filamentous fungal cell is not anAspergillus cell.

In certain embodiments, the filamentous fungal cell is selected from thegroup consisting of Trichoderma (T. reesei), Neurospora (N. crassa),Penicillium (P. chrysogenum), Aspergillus (A. nidulans, A. niger and A.oryzae), Myceliophthora (M. thermophila) and Chrysosporium (C.lucknowense).

In certain embodiments, the filamentous fungal cell is a Trichodermafungal cell. Trichoderma fungal cells of the present disclosure may bederived from a wild-type Trichoderma strain or a mutant thereof.Examples of suitable Trichoderma fungal cells include, withoutlimitation, Trichoderma harzianum, Trichoderma koningii, Trichodermalongibrachiatum, Trichoderma reesei, Trichoderma atroviride, Trichodermavirens, Trichoderma viride; and alternative sexual form thereof (i.e.,Hypocrea).

General methods to disrupt genes of and cultivate filamentous fungalcells are disclosed, for example, for Penicillium, in Kopke et al.(2010) Application of the Saccharomyces cerevisiae FLP/FRT recombinationsystem in filamentous fungi for marker recycling and construction ofknockout strains devoid of heterologous genes. Appl Environ Microbiol.76(14):4664-74. doi: 10.1128/AEM.00670-10, for Aspergillus, in Maruyamaand Kitamoto (2011), Targeted Gene Disruption in Koji Mold Aspergillusoryzae, in James A. Williams (ed.), Strain Engineering: Methods andProtocols, Methods in Molecular Biology, vol. 765, DOI10.1007/978-1-61779-197-0_27; for Neurospora, in Collopy et al. (2010)High-throughput construction of gene deletion cassettes for generationof Neurospora crassa knockout strains. Methods Mol Biol. 2010;638:33-40. doi: 10.1007/978-1-60761-611-5_3; and for Myceliophthora orChrysosporium PCT/NL2010/000045 and PCT/EP98/06496.

Filamentous Fungal Cell Components

Certain aspects of the present disclosure relate to filamentous fungalcells having reduced or no detectable activity of at least threeproteases and having a recombinant polynucleotide encoding aheterologous polypeptide that is produced at increased levels, forexample at least two-fold increased levels. Other aspects of the presentdisclosure relate to Trichoderma or closely related species fungal cellsthat has reduced or no detectable protease activity of at least two,three or four proteases selected from pep9, amp1, amp2 and sep1. Otheraspects of the present disclosure relate to Trichoderma or closelyrelated species fungal cells that has reduced or no detectable proteaseactivity in one or more of the following proteases selected from pep9,amp1, amp2, mp1, mp2, mp3, mp4, mp5 and sep1. Other aspects of thepresent disclosure relate to Trichoderma or closely related speciesfungal cells that has reduced or no detectable protease activity of atleast two, three or four proteases selected from proteases selected frompep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1, slp1, slp2,slp3, slp7, gap1, and gap2, where the cell further contains arecombinant polynucleotide encoding a mammalian polypeptide produced ata level of at least 2-fold higher than the production level of thepolypeptide in a corresponding parental Trichoderma fungal cell. Incertain embodiments, the filamentous fungal cells or Trichoderma fungalcells have reduced or no activity of at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,at least eleven, at least twelve, or more proteases.

Reduced Expression of Proteases

The reduced activity of the at least three proteases in filamentousfungal cells or Trichoderma fungal cells of the present disclosure canbe the result of reduced or eliminated expression of the proteases. Insome embodiments, the reduced or eliminated expression of the at leastthree proteases is the result of a modification to the catalytic domain,the coding region, or a control sequence required for expression of thecoding region of the genes encoding each of the proteases. In otherembodiments, the reduced or eliminated expression of the proteases isthe result of introducing, substituting, and/or removing one or morenucleotides in the genes, or a control sequence thereof required for thetranscription or translation of the genes encoding each of theproteases. In some embodiments, the reduced or eliminated expression ofthe proteases is the result of introducing, recombining, or replacingthe endogenous promoter, or part thereof, of the protease with aheterologous promoter. The “heterologous promoter” herein means anoperatively linked promoter DNA sequence which is non-native for theprotease. In some embodiments, the heterologous promoter reducesprotease activity compared to the activity in the corresponding parentalfilamentous fungal cell in which the protease is expressed by itsendogenous promoter. If deleting the protease affects growth,sporulation or function of the fungus, the heterologous promoter beselected in such a way that the protease is expressed, for example,during essential phase of the cell, for example sporulation, but theprotease expression is reduced or eliminated during production ofheterologous protein compared to the corresponding parental strain inwhich the protease is expressed by its endotenous promoter.

In some embodiments, the heterologous promoter is a constitutivepromoter or an inducible promoter. In some embodiments, the heterologouspromoter is selected from the group of flavin containing monooxygenasegene (Tre76230), the RNA polymerase gene (Tre49048), and the slp8protease gene (Tre58698).

In some embodiments, the reduced or eliminated protease with aheterologous promoter is slp2, e.g., slp2 in Trichoderma fungal cell orclosely related species. In some specific embodiments, the endogenouspromoter of slp2, e.g. in Trichoderma or closely related species, isreplaced with a heterologous promoter selected from the group of flavincontaining monooxygenase gene (Tre76230), the RNA polymerase gene(Tre49048), and the slp8 protease gene (Tre58698). More details aregiven in Example 41.

In some embodiments, a filamentous fungal cell of the inventioncomprises at least one endogenous protease having a heterologouspromoter and having reduced or no protease activity, and a recombinantpolynucleotide encoding a heterologous polypeptide, wherein the cell hasreduced or no protease activity in one or more of following proteases:pep9, amp1, amp2, mp1, mp2, mp3, mp4, mp5 and sep1, and, optionally oneor more additional proteases selected from pep1, pep2, pep3, pep4, pep5,pep8, pep11, pep12, tsp1, slp1, slp2, slp3, slp7, gap1, and gap2.

In further embodiments, the reduced or eliminated expression of theproteases is the result of inserting into the genes encoding each of theproteases disruptive nucleic acid constructs each containing a nucleicacid fragment homologous to each of the genes that will create aduplication of the region of homology and incorporate construct DNAbetween the duplicated regions. In other embodiments, the reduced oreliminated expression of the proteases is the result of gene conversionof the genes encoding each of the proteases. In still other embodiments,the reduced or eliminated expression of the proteases is the result ofby anti-sense polynucleotides or RNAi constructs that are specific forthe each of the genes encoding each of the proteases. In one embodiment,an RNAi construct is specific for a gene encoding an aspartic proteasesuch as a pep-type protease, a trypsin-like serine proteases such as atsp1, a glutamic protease such as a gap-type protease, a subtilisinprotease such as a sip-type protease, or a sedolisin protease such as atpp1 or a slp7 protease. In one embodiment, an RNAi construct isspecific for the gene encoding a sip-type protease. In one embodiment,an RNAi construct is specific for the gene encoding slp2, slp3, slp5 orslp6. In one embodiment, an RNAi construct is specific for two or moreproteases. In one embodiment, two or more proteases are any one of thepep-type proteases, any one of the trypsin-like serine proteasess, anyone of the sip-type proteases, any one of the gap-type proteases, anyone of the metalloproteases and/or any one of the sedolisin proteases.In one embodiment, two or more proteases are slp2, slp3, slp5 and/orslp6. In one embodiment, RNAi construct comprises any one of nucleicacid sequences of Table 22.2.

In some embodiments, the genes encoding the proteases each contain amutation that reduces or eliminates the corresponding protease activity.In other embodiments, the mutation reduces or eliminates the expressionof each of the proteases. In further embodiments, the mutation is aknock-out mutation, a truncation mutation, a point mutation, a missensemutation, a substitution mutation, a frameshift mutation, an insertionmutation, a duplication mutation, an amplification mutation, atranslocation mutation, an inversion mutation that reduces or eliminatesthe corresponding protease activity.

In some embodiments, the mutation is a deletion of the protease encodinggene. In other embodiments, the mutation is a deletion of the portion ofthe protease encoding gene encoding the catalytic domain of theprotease. In still other embodiments, the mutation is point mutation inthe portion of the protease encoding gene encoding the catalytic domainof the protease.

Another method to disrupt protease genes of filamentous fungal cellsinclude CRISPR-CAS system, or clustered regularly interspaced shortpalindromic repeats. CRISPR-Cas system is a novel technique of geneediting (silencing, enhancing or changing specific genes). By insertinga plasmid containing cas9 genes and specifically designed CRISPRs, theorganism's genome can be cut at any desired location. Cas9 geneoriginates from the type II bacterial CRISPR system of Streptococcuspyogenes. Gene product, CAS9 nuclease, complexes with a specific genometargeting CRISPR guideRNA and has high site specificity of the DNAcutting activity. It has been shown recently that CAS9 can function asan RNA-guided endonuclease in various heterologous organisms (Mali etal. 2013: Rna guided human genome engineering via Cas9. Science339:823-826; Cong et al 2013: Multiplex genome engineering usingCRISPR-Cas systems. Science 339:819-823; Jiang et al 2013: RNA-guidedediting of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol31:233-239; Jinek et al. 2013: RNA programmed genome editing in humancells. eLife 2:e00471; Hwang et al. 2013: Efficient genome editing inzebrafish using a CRISPR-Cas system. Nat Biotech 31:227-279. DiCarlo etal 2013: Genome engineering in Saccharomyces cerevisiae using CRISPR-Cassystems. NAR 41:4336-4343).

GuideRNA synthesis have been usually carried out from promoterstranscribed by RNA polymerase III, most commonly used being SNR52 snoRNApromoter in yeasts and U3/U6 snoRNA promoters in plants and animals.Promoters transcribed by RNA polymerase II have been considered to beunsuitable for guideRNA synthesis because of the posttranscriptionalmodifications, 5′capping, 5′/3′ UTR's and poly A tailing. However, ithas been recently demonstrated that RNA polymerase II type promoters canbe used if the guideRNA sequence is flanked with self-processingribozyme sequences. Primary transcript then undergoes self-catalyzedcleavage and generates desired gRNA sequence (Gao and Zhao 2014: Selfprocessing of ribozyme-flanked RNAs into guide RNA's in vitro and invivo for CRISPR-mediated genome editing. Journal of Integrative PlantBiology e-publication ahead of print; March 2014).

Example 21 exemplifies methods to disrupt various protease encodinggenes that affect and/or hinder efficient production of heterologousproteins in T. reesei. GuideRNA sequences as shown in Table 21.1 andtheir use for disrupting corresponding protease genes in Trichodermacells are also part of the invention.

Combinations of Protease Genes

The filamentous fungal cells or Trichoderma fungal cells of the presentdisclosure may contain at least three, at least four, at least five, atleast six, at least seven, at least eight, at least nine, at least ten,or more aspartic proteases, trypsin-like serine proteases, subtilisinproteases, glutamic proteases, and metalloproteases. In certainembodiments, the proteases are encoded by pep-type protease genes,gap-type protease genes, sip-type proteases genes, amp-type protease,sep-type protease and mp-type proteases. In some embodiments, thepep-type protease genes are selected from pep1, pep2, pep3, pep4, pep5,pep6, pep8, pep9, pep10, pep11, and pep12, pep13, pep14, pep16. In otherembodiments, the gap-type protease genes are selected from gap1, andgap2. In other embodiments, the gap-type protease genes are selectedfrom gap1, gap2, gap3 or gap4. In further embodiments, the sip-typeproteases genes are selected from slp1, slp2, slp3, and slp7; or areselected from slp1, slp2, slp3, slp5, slp6, slp7, and slp8, or areselected from slp1, slp2, slp3, slp5, slp6, slp7, and slp8 and slp57433,slp35726, slp60791, or slp109276. In certain preferred embodiments, thesip-type proteases gene is slp1. In certain embodiments, the amp-typeproteases are selected from amp1 and amp2. In further embodiments, thesep-type proteases are selected from sep1, sed2, sed3, and sed5. Infurther embodiments, the mp-type metalloproteases are selected from mp1,mp2, mp3, mp4 and mp5. In other embodiments, the proteases are encodedby genes selected from pep9, amp1, amp2, sep1, pep1, pep2, pep3, pep4,pep5, pep7, pep8, pep11, pep12, tsp1, slp1, slp2, slp3, slp5, slp6,slp7, slp8, gap1, gap2, and tpp1. In some embodiments, the filamentousfungal cell, for example, a Trichoderma cell has reduced or noexpression levels of at least two, three or at least four protease of afirst group of protease selected from pep9, amp1, amp2 and sep1,optionally in combination with a second group of protease encoding genesselected from pep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1,slp1, slp2, slp3, slp7, gap1, and gap2. In some embodiments, thefilamentous fungal cell, for example, a Trichoderma cell has reduced orno expression levels in one or more of the following protease selectedfrom pep9, amp1, amp2, mp1, mp2, mp3, mp4, mp5 and sep1, optionally incombination with a second group of protease encoding genes selected frompep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1, slp1, slp2,slp3, slp7, gap1, and gap2. In certain embodiments, the filamentousfungal cell, for example a Trichoderma cell, has reduced or noexpression levels of at least three protease encoding genes selectedfrom pep1, tsp1, and slp1. In other embodiments, the filamentous fungalcell, or Trichoderma cell, has reduced or no expression levels of atleast three protease encoding genes selected from gap1, slp1 and pep1.In some embodiments, the filamentous fungal cell, for example, aTrichoderma cell has reduced or no expression levels of proteaseencoding genes slp2, pep1, and gap1. In some embodiments, thefilamentous fungal cell, for example, a Trichoderma cell has reduced orno expression levels of protease encoding genes slp2, pep1, gap1, andpep4. In some embodiments, the filamentous fungal cell, for example, aTrichoderma cell has reduced or no expression levels of proteaseencoding genes slp2, pep1, gap1, pep4, and slp1. In some embodiments,the filamentous fungal cell, for example, a Trichoderma cell has reducedor no expression levels of protease encoding genes slp2, pep1, gap1,pep4, slp1, and slp3. In some embodiments, the filamentous fungal cell,for example, a Trichoderma cell has reduced or no expression levels ofprotease encoding genes slp2, pep1, gap1, pep4, slp1, slp3, and pep3. Insome embodiments, the filamentous fungal cell, for example, aTrichoderma cell has reduced or no expression levels of proteaseencoding genes slp2, pep1, gap1, pep4, slp1, slp3, pep3, and pep2.

In some embodiments, the filamentous fungal cell, for example, aTrichoderma cell has reduced or no expression levels of proteaseencoding genes slp2, pep1, gap1, pep4, slp1, slp3, pep3, pep2, and pep5.In some embodiments, the filamentous fungal cell, for example, aTrichoderma cell has reduced or no expression levels of proteaseencoding genes slp2, pep1, gap1, pep4, slp1, slp3, pep3, pep2, pep5, andtsp1. In some embodiments, the filamentous fungal cell, for example, aTrichoderma cell has reduced or no expression levels of proteaseencoding genes slp2, pep1, gap1, pep4, slp1, slp3, pep3, pep2, pep5,tsp1, and slp7. In some embodiments, the cell, for example a Trichodermacell, has reduced or no protease activity in at least twelve proteases,each of the genes encoding the twelve proteases comprises a mutationthat reduces or eliminates the corresponding protease activity, and thetwelve proteases are pep1, slp1, gap1, gap2, pep4, pep3, pep5, pep2,sep1, slp8, amp2, slp7. In other embodiments, the cell, for example aTrichoderma cell has either

-   -   (i) reduced or no protease activity in at least thirteen        proteases, each of the genes encoding the thirteen proteases        comprises a mutation that reduces or eliminates the        corresponding protease activity, and the thirteen proteases are        either        -   pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1,            slp8, amp2, pep9;        -   pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1,            slp8, amp2, slp7; or        -   pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1,            slp8, amp2, slp3;    -   (ii) the cell has reduced or no protease activity in at least        fourteen proteases, each of the genes encoding the fourteen        proteases comprises a mutation that reduces or eliminates the        corresponding protease activity, and the fourteen proteases are        pep1 tsp1 slp1 gap1 gap2 pep4 pep3 pep5 pep2 sep1 slp8 amp2 pep9        slp2;    -   (iii) the cell has reduced or no protease activity in at least        fifteen proteases, each of the genes encoding the fifteen        proteases comprises a mutation that reduces or eliminates the        corresponding protease activity, and the fifteen proteases are        either        -   pep1 tsp1 slp1 gap1 gap2 pep4 pep3 pep5 pep2 sep1 slp8 amp2            pep9 slp2 mp1; or,        -   pep1 tsp1 slp1 gap1 gap2 pep4 pep3 pep5 pep2 sep1 slp8 amp2            pep9 slp2 mp5.

In other embodiments, the filamentous fungal cell of the invention, forexample a Trichoderma cell, has reduced or no protease activity in atleast sixteen, at least seventeen, at least eighteen, at least nineteen,or at least twenty or more proteases, with 13, 14, or 15 mutations inthe above recited proteases, and further comprising one or moreadditional mutations, each additional mutation reduces or eliminates acorresponding additional protease activity, said additional proteasebeing selected from the group consisting of

-   -   an aspartic protease pep6, pep1 0, pep13, pep14, or pep16;    -   slp like protease slp57433, slp35726, slp60791, or slp109276;    -   gap like protease gap3 or gap4;    -   sedolisin like protease sed2, sed3, or sed5;    -   Group A protease selected from the group of protease65735,        protease77577, protease81087, protease56920, protease122083,        protease79485, protease120998, or protease61127;    -   Group B protease selected from the group of protease21659,        protease58387, protease75159, protease56853, or protease64193;    -   Group C protease selected from the group of protease82452,        protease80762, protease21668, protease81115, protease82141,        protease23475;    -   Group D protease selected from the group of protease121890,        protease22718, protease47127, protease61912, protease80843,        protease66608, protease72612, protease40199; or    -   Group E protease selected from the group of protease22210,        protease111694, protease82577.

In some embodiments, the filamentous fungal cell, for example, aTrichoderma cell has reduced or no expression levels of proteaseencoding genes slp2, pep1, gap1, pep4, slp1, slp3, pep3, pep2, pep5,tsp1, slp7, and slp8. In some embodiments, the filamentous fungal cell,for example, a Trichoderma cell has reduced or no expression levels ofprotease encoding genes slp2, pep1, gap1, pep4, slp1, slp3, pep3, pep2,pep5, tsp1, slp7, slp8, and gap2.

In certain embodiments, the filamentous fungal cell has at least three,at least four, at least five, at least six, at least seven, at leasteight, at least nine, at least ten, or more proteases with reducedprotease activity, wherein the corresponding proteases with wild typeactivity each have an amino acid sequence that is at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% identical to theamino acid sequences of SEQ ID NOs: 1-16; 17-36; 37-57; 58-65; 66-81;82-97; 98-117; 118-128; 129-144; 166-181; 182-185; 491-588, SEQ ID NOs750-805 or SEQ ID NOs 875-923. In embodiments where the filamentousfungal cell is a Trichoderma fungal cell with reduced protease activityin one or more proteases, wherein the corresponding proteases with wildtype activity each have an amino acid sequence that is at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100% identicalto the amino acid sequences of SEQ ID NOs: 1, 17, 37, 58, 66, 82, 98,118, 129, 166, 182, 507, 522, 530, 750, 759, 775, SEQ ID NO:786, or SEQID NOs 875-923.

Heterologous Polypeptides

The filamentous fungal cells or Trichoderma fungal cells of the presentdisclosure contain a recombinant polynucleotide encoding a heterologouspolypeptide. In certain embodiments, the heterologous polypeptide is amammalian polypeptide. In other embodiments, heterologous polypeptide isa non-mammalian polypeptide.

In embodiments where the filamentous fungal cell contains a recombinantpolynucleotide encoding a mammalian polypeptide, the mammalianpolypeptide can be a non-glycosylated mammalian polypeptide, aglycosylated mammalian polypeptide, or combinations thereof, including,without limitation, an immunoglobulin, an antibody, a growth factor, andan interferon. In some embodiments, the mammalian polypeptide is animmunoglobulin or antibody. In embodiments where the filamentous fungalcell contains a recombinant polynucleotide encoding an immunoglobulin orantibody, the filamentous fungal cell, for example, a Trichoderma fungalcell may have reduced or no expression of at least two, three or atleast four protease encoding genes selected from pep9, amp1, amp2 andsep1, optionally in combination with at least three or four proteasesselected from pep1, pep3, pep4, pep8, pep11, pep12, tsp1, slp1, slp2,slp7, gap1, and gap2. In some embodiments where the filamentous fungalcell contains a recombinant polynucleotide encoding an immunoglobulin orantibody, for example, a Trichoderma cell, said cell has reduced or noexpression levels in one or more of the following protease selected frompep9, amp1, amp2, mp1, mp2, mp3, mp4, mp5 and sep1, optionally incombination with one or more additional proteases encoding genesselected from pep1, pep2, pep3, pep4, pep5, pep8, pep11, pep12, tsp1,slp1, slp2, slp3, slp7, gap1, and gap2. In certain preferredembodiments, the cell, for example a Trichoderma fungal cell, contains arecombinant polynucleotide encoding an immunoglobulin or antibody andhas reduced or no expression of the protease encoding genes pep1, tsp1,slp1, and gap1. In other embodiments, the cell contains a recombinantpolynucleotide encoding an immunoglobulin or antibody and has reducedexpression of the protease encoding genes pep1, tsp1, slp1, gap1, andpep4. In other embodiments, the cell contains a recombinantpolynucleotide encoding an immunoglobulin or antibody and has reducedexpression of the protease encoding genes slp1, slp2, and slp3. In otherembodiments, the cell contains a recombinant polynucleotide encoding animmunoglobulin or antibody and has reduced expression of the proteaseencoding genes slp1, slp2, slp3, and tsp1. In other embodiments, thecell contains a recombinant polynucleotide encoding an immunoglobulin orantibody and has reduced expression of the protease encoding genes slp1,slp2, slp3, tsp1, and pep1. In other embodiments, the cell contains arecombinant polynucleotide encoding an immunoglobulin or antibody andhas reduced expression of the protease encoding genes slp1, slp2, slp3,tsp1, pep1, and gap1. In other embodiments, the cell contains arecombinant polynucleotide encoding an immunoglobulin or antibody andhas reduced expression of the protease encoding genes slp1, slp2, slp3,tsp1, pep1, gap1, and pep4. In other embodiments, the cell contains arecombinant polynucleotide encoding an immunoglobulin or antibody andhas reduced expression of the protease encoding genes slp1, slp2, slp3,tsp1, pep1, gap1, pep4, and pep3. In other embodiments, the cellcontains a recombinant polynucleotide encoding an immunoglobulin orantibody and has reduced expression of the protease encoding genes slp1,slp2, slp3, tsp1, pep1, gap1, pep4, pep3, and pep2. In otherembodiments, the cell contains a recombinant polynucleotide encoding animmunoglobulin or antibody and has reduced expression of the proteaseencoding genes slp1, slp2, slp3, tsp1, pep1, gap1, pep4, pep3, pep2, andpep5. In some embodiments, the cell, for example a Trichoderma cell,contains a recombinant polynucleotide encoding an immunoglobulin orantibody and has reduced or no protease activity in at least twelveproteases, each of the genes encoding the twelve proteases comprises amutation that reduces or eliminates the corresponding protease activity,and the twelve proteases are pep1, slp1, gap1, gap2, pep4, pep3, pep5,pep2, sep1, slp8, amp2, slp7. In other embodiments, the cell, forexample a Trichoderma cell, contains a recombinant polynucleotideencoding an immunoglobulin or antibody and has either

-   -   (i) reduced or no protease activity in at least thirteen        proteases, each of the genes encoding the thirteen proteases        comprises a mutation that reduces or eliminates the        corresponding protease activity, and the thirteen proteases are        either        -   pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1,            slp8, amp2, pep9;        -   pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1,            slp8, amp2, slp7;        -   pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1,            slp8, amp2, slp3;    -   (ii) reduced or no protease activity in at least fourteen        proteases, each of the genes encoding the fourteen proteases        comprises a mutation that reduces or eliminates the        corresponding protease activity, and the fourteen proteases are        pep1 tsp1 slp1 gap1 gap2 pep4 pep3 pep5 pep2 sep1 slp8 amp2 pep9        slp2; or,    -   (iii) reduced or no protease activity in at least fifteen        proteases, each of the genes encoding the fifteen proteases        comprises a mutation that reduces or eliminates the        corresponding protease activity, and the fifteen proteases are        either        -   pep1 tsp1 slp1 gap1 gap2 pep4 pep3 pep5 pep2 sep1 slp8 amp2            pep9 slp2 mp1; or,        -   pep1 tsp1 slp1 gap1 gap2 pep4 pep3 pep5 pep2 sep1 slp8 amp2            pep9 slp2 mp5.

In other embodiments, the filamentous fungal cell, for example aTrichoderma cell, contains a recombinant polynucleotide encoding animmunoglobulin or antibody and has reduced or no protease activity in atleast sixteen, at least seventeen, at least eighteen, at least nineteen,or at least twenty or more proteases, with 13, 14 or 15 mutations in theabove recited proteases, and further comprising one or more additionalmutation, each additional mutation reduces or eliminates thecorresponding additional protease activity, and said at least additionalprotease being selected from the group consisting of

-   -   an aspartic protease pep6, pep10, pep13, pep14, or pep16;    -   slp like protease slp57433, slp35726, slp60791, or slp109276;    -   gap like protease gap3 or gap4;    -   sedolisin like protease sed2, sed3, or sed5;    -   Group A protease selected from the group of protease65735,        protease77577, protease81087, protease56920, protease122083,        protease79485, protease120998, or protease61127;    -   Group B protease selected from the group of protease21659,        protease58387, protease75159, protease56853, or protease64193;    -   Group C protease selected from the group of protease82452,        protease80762, protease21668, protease81115, protease82141,        protease23475;    -   Group D protease selected from the group of protease121890,        protease22718, protease47127, protease61912, protease80843,        protease66608, protease72612, protease40199; or    -   Group E protease selected from the group of protease22210,        protease111694, protease82577.

In other embodiments, the filamentous fungal cell contains a recombinantpolynucleotide encoding a growth factor, interferon, cytokine, orinterleukin. In embodiments where the filamentous fungal cell, forexample a Trichoderma fungal cell contains a recombinant polynucleotideencoding a growth factor, interferon, cytokine, human serum albumin, orinterleukin, the filamentous fungal cell may have reduced or noexpression of at least two, three or four proteases selected from pep9,amp1, amp2 and sep1, optionally in combination with at least three orfour proteases selected from pep1, pep3, pep4, pep8, pep11, pep12, tsp1,slp1, slp2, slp7, gap1, and gap2 or at least three or at least fourprotease encoding genes selected from pep1, pep2, pep3, pep4, pep5,pep8, gap1, gap2, slp1, slp2, slp7, and tsp1.

In some embodiments where the filamentous fungal cell contains arecombinant polynucleotide encoding a growth factor, interferon,cytokine, human serum albumin, or interleukin, for example, aTrichoderma cell, said cell has reduced or no expression levels in oneor more of the following protease selected from pep9, amp1, amp2, mp1,mp2, mp3, mp4, mp5 and sep1, optionally in combination with a secondgroup of protease encoding genes selected from pep1, pep2, pep3, pep4,pep5, pep8, pep11, pep12, tsp1, slp1, slp2, slp3, slp7, gap1, and gap2.In certain embodiments, the cell contains a recombinant polynucleotideencoding a growth factor, interferon, cytokine, human serum albumin, orinterleukin and has reduced expression of the protease encoding genespep1, tsp1, slp1, gap1, and gap2.

In certain embodiments, the cell contains a recombinant polynucleotideencoding a growth factor, interferon, cytokine, human serum albumin, orinterleukin and has reduced expression of the protease encoding genesslp1, slp2, pep1, gap1, pep4, slp7, pep2, pep3, pep5, tsp1, and gap2. Inother embodiments, the cell, for example a Trichoderma fungal cell,contains a recombinant polynucleotide encoding a growth factor,interferon, cytokine, human serum albumin, or interleukin and hasreduced expression of the protease encoding genes pep1, tsp1, slp1,gap1, gap2, and pep4. In a further embodiment, the cell contains arecombinant polynucleotide encoding a growth factor, and has reducedexpression of a pep-type protease genes are selected from pep1, pep2,pep3, pep4, and pep5.

In certain preferred embodiments, the growth factor is IGF-1 or theinterferon is interferon-α 2b. In certain embodiments, the cell containsa recombinant polynucleotide encoding a growth factor, interferon,cytokine, human serum albumin, or interleukin and has reduced expressionof the protease encoding genes pep1, gap1, and pep4. In certainembodiments, the cell contains a recombinant polynucleotide encoding agrowth factor, interferon, cytokine, human serum albumin, or interleukinand has reduced expression of the protease encoding genes pep1, gap1,pep4, and slp7. In certain embodiments, the cell contains a recombinantpolynucleotide encoding a growth factor, interferon, cytokine, humanserum albumin, or interleukin and has reduced expression of the proteaseencoding genes pep1, gap1, pep4, slp7, and slp2. In certain embodiments,the cell contains a recombinant polynucleotide encoding a growth factor,interferon, cytokine, human serum albumin, or interleukin and hasreduced expression of the protease encoding genes pep1, gap1, pep4,slp7, slp2, and pep2.

In certain embodiments, the cell contains a recombinant polynucleotideencoding a growth factor, interferon, cytokine, human serum albumin, orinterleukin and has reduced expression of the protease encoding genespep1, gap1, pep4, slp7, slp2, pep2, and pep3. In certain embodiments,the cell contains a recombinant polynucleotide encoding a growth factor,interferon, cytokine, human serum albumin, or interleukin and hasreduced expression of the protease encoding genes pep1, gap1, pep4,slp7, slp2, pep2, pep3, and pep5. In certain embodiments, the cellcontains a recombinant polynucleotide encoding a growth factor,interferon, cytokine, human serum albumin, or interleukin and hasreduced expression of the protease encoding genes pep1, gap1, pep4,slp7, slp2, pep2, pep3, pep5, and slp1. In certain embodiments, the cellcontains a recombinant polynucleotide encoding a growth factor,interferon, cytokine, human serum albumin, or interleukin and hasreduced expression of the protease encoding genes pep1, gap1, pep4,slp7, slp2, pep2, pep3, pep5, slp1, and tsp1.

In some embodiments, the cell, for example a Trichoderma cell, containsa recombinant polynucleotide encoding a growth factor, interferon,cytokine, human serum albumin, or interleukin and has reduced or noprotease activity in at least twelve proteases, each of the genesencoding the twelve proteases comprises a mutation that reduces oreliminates the corresponding protease activity, and the twelve proteasesare pep1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1, slp8, amp2,slp7. In other embodiments, the cell, for example a Trichoderma cell,contains a recombinant polynucleotide encoding a growth factor,interferon, cytokine, human serum albumin, or interleukin and has either

-   -   (i) reduced or no protease activity in at least thirteen        proteases, each of the genes encoding the thirteen proteases        comprises a mutation that reduces or eliminates the        corresponding protease activity, and the thirteen proteases are        either        -   pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1,            slp8, amp2, pep9;        -   pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1,            slp8, amp2, slp7;        -   pep1, tsp1, slp1, gap1, gap2, pep4, pep3, pep5, pep2, sep1,            slp8, amp2, slp3;    -   (ii) reduced or no protease activity in at least fourteen        proteases, each of the genes encoding the fourteen proteases        comprises a mutation that reduces or eliminates the        corresponding protease activity, and the fourteen proteases are        pep1 tsp1 slp1 gap1 gap2 pep4 pep3 pep5 pep2 sep1 slp8 amp2 pep9        slp2; or,    -   (iii) reduced or no protease activity in at least fifteen        proteases, each of the genes encoding the fifteen proteases        comprises a mutation that reduces or eliminates the        corresponding protease activity, and the fifteen proteases are        either        -   pep1 tsp1 slp1 gap1 gap2 pep4 pep3 pep5 pep2 sep1 slp8 amp2            pep9 slp2 mp1; or,        -   pep1 tsp1 slp1 gap1 gap2 pep4 pep3 pep5 pep2 sep1 slp8 amp2            pep9 slp2 mp5.

In other embodiments, the filamentous fungal cell, for example aTrichoderma cell, contains a recombinant polynucleotide encoding growthfactor, interferon, cytokine, human serum albumin, or interleukin andhas reduced or no protease activity in at least sixteen, at leastseventeen, at least eighteen, at least nineteen, or at least twenty ormore proteases, with mutations in the above recited 13, 14 or 15proteases, and further comprising one or more additional mutation, eachadditional mutation reduces or eliminates the corresponding additionalprotease activity, and said at least additional protease being selectedfrom the group consisting of

-   -   an aspartic protease pep6, pep1 0, pep13, pep14, or pep16;    -   slp like protease slp57433, slp35726, slp60791, or slp109276;    -   gap like protease gap3 or gap4;    -   sedolisin like protease sed2, sed3, or sed5;    -   Group A protease selected from the group of protease65735,        protease77577, protease81087, protease56920, protease122083,        protease79485, protease120998, or protease61127;    -   Group B protease selected from the group of protease21659,        protease58387, protease75159, protease56853, or protease64193;    -   Group C protease selected from the group of protease82452,        protease80762, protease21668, protease81115, protease82141,        protease23475;    -   Group D protease selected from the group of protease121890,        protease22718, protease47127, protease61912, protease80843,        protease66608, protease72612, protease40199; or    -   Group E protease selected from the group of protease22210,        protease111694, protease82577.

In certain embodiments, the mammalian polypeptide is produced at a levelthat is at least 3-fold, at least 4-fold, at least 5-fold, at least6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least70-fold, at least 75-fold, at least 80-fold, at least 90-fold, at least100-fold, or a greater fold higher than the production level of thepolypeptide in a corresponding parental filamentous fungal cell withoutthe reduced protease activity. In other embodiments, the mammalianpolypeptide is produced in a full length version at a level higher thanthe production level of the full-length version of the polypeptide in acorresponding parental filamentous fungal cell.

In embodiments where the filamentous fungal cell contains a recombinantpolynucleotide encoding a non-mammalian polypeptide, the non-mammalianpolypeptide may be an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase,deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase,glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase,lipase, mutanase, oxidase, pectinolytic enzyme, peroxidase,phospholipase, phytase, polyphenoloxidase, proteolytic enzyme,ribonuclease, transglutaminase or xylanase. In embodiments where thefilamentous fungal cell contains a recombinant polynucleotide encoding anon-mammalian polypeptide, the filamentous fungal cell may have reducedor no detectable expression of at least two, three or four proteasesselected from pep9, amp1, amp2 mp1, mp2, mp3, mp4, mp5 and sep1,optionally in combination with three, at least four, at least five, orat least six protease encoding genes selected from pep1, pep2, pep2,pep4, pep5, pep8, pep11, pep12, tsp1, slp1, slp2, slp3, slp7, gap1, andgap2. In certain embodiments, the non-mammalian polypeptide is producedat a level that is at least 3-fold, at least 4-fold, at least 5-fold, atleast 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, atleast 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, atleast 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, atleast 70-fold, at least 75-fold, at least 80-fold, at least 90-fold, atleast 100-fold, or a greater fold higher than the production level ofthe polypeptide in a corresponding parental filamentous fungal cell. Inother embodiments, the non-mammalian polypeptide is produced in a fulllength version at a level higher than the production level of thefull-length version of the polypeptide in a corresponding parentalfilamentous fungal cell.

Reduced Activity of Additional Proteases

In some embodiments, the filamentous fungal cells or Trichoderma fungalcells of the present disclosure also have reduced activity of one ormore additional proteases. In certain embodiments, the expression levelof the one or more additional proteases is reduced. In certain preferredembodiments, genes encoding the one or more additional proteases eachcomprise a mutation that reduces the corresponding protease activity.The one or more additional protease encoding genes may be pep7, tpp1,gap2, slp3, slp5, slp6, slp7, slp8, or a gene encoding protease selectedfrom the group consisting of

-   -   an aspartic protease pep6, pep1 0, pep13, pep14, or pep16;    -   slp like protease slp57433, slp35726, slp60791, or slp109276;    -   gap like protease gap3 or gap4;    -   sedolisin like protease sed2, sed3, or sed5;    -   Group A protease selected from the group of protease65735,        protease77577, protease81087, protease56920, protease122083,        protease79485, protease120998, or protease61127;    -   Group B protease selected from the group of protease21659,        protease58387, protease75159, protease56853, or protease64193;    -   Group C protease selected from the group of protease82452,        protease80762, protease21668, protease81115, protease82141,        protease23475;    -   Group D protease selected from the group of protease121890,        protease22718, protease47127, protease61912, protease80843,        protease66608, protease72612, protease40199; or    -   Group E protease selected from the group of protease22210,        protease111694, protease82577.

In certain embodiments, when the filamentous fungal cells is anAspergillus cell, the total protease activity is reduced to 50% or lessof the total protease activity in the corresponding parental Aspergilluscell in which the proteases do not have reduced activity.

In certain embodiments, total protease activity is reduced in the cellof the present disclosure, for example a Trichoderma cell, to 49% orless, 31% or less, 13% or less, 10% or less, 6.3% or less, or 5.5% orless, of the total protease activity in the corresponding parentalfilamentous fungal cell in which the proteases do not have reducedactivity.

Additional Recombinant Modifications

In certain embodiments, the filamentous fungal cells or Trichodermafungal cells of the present disclosure also have reduced activity of adolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase.Dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase (EC2.4.1.130) transfers an alpha-D-mannosyl residue from dolichyl-phosphateD-mannose into a membrane lipid-linked oligosaccharide. Typically, thedolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase enzyme isencoded by an alg3 gene. Thus, in certain embodiments, the filamentousfungal cell has reduced activity of ALG3, which is the activity encodedby the alg3 gene. In some embodiments, the alg3 gene contains a mutationthat reduces the corresponding ALG3 activity. In certain embodiments,the alg3 gene is deleted from the filamentous fungal cell.

In other embodiments, the filamentous fungal cells or Trichoderma fungalcells of the present disclosure further contain a polynucleotideencoding an α-1,2-mannosidase. The polynucleotide encoding theα-1,2-mannosidase may be endogenous in the host cell, or it may beheterologous to the host cell. These polynucleotides are especiallyuseful for a filamentous fungal cell expressing high-mannose glycanstransferred from the Golgi to the ER without effectiveexo-α-2-mannosidase cleavage. The α-1,2-mannosidase may be a mannosidaseI type enzyme belonging to the glycoside hydrolase family 47(cazy.org/GH47_all.html). In certain embodiments the α-1,2-mannosidaseis an enzyme listed at cazy.org/GH47_characterized.html. In particular,the α-1,2-mannosidase may be an ER-type enzyme that cleavesglycoproteins such as enzymes in the subfamily of ER α-mannosidase I EC3.2.1.113 enzymes. Examples of such enzymes include humanα-2-mannosidase 1B (AAC26169), a combination of mammalian ERmannosidases, or a filamentous fungal enzyme such as α-1,2-mannosidase(MDS1) (T. reesei AAF34579; Maras M et al J Biotech. 77, 2000, 255). ForER/Golgi expression the catalytic domain of the mannosidase is typicallyfused with a targeting peptide, such as HDEL, KDEL, or part of an ER orearly Golgi protein, or expressed with an endogenous ER targetingstructures of an animal or plant mannosidase I enzyme, see, for example,Callewaert et al. 2001 Use of HDEL-tagged Trichoderma reesei mannosyloligosaccharide 1,2-α-D-mannosidase for N-glycan engineering in Pichiapastoris. FEBS Lett 503: 173-178.

In further embodiments, the filamentous fungal cells or Trichodermafungal cells of the present disclosure also contain anN-acetylglucosaminyltransferase I catalytic domain and anN-acetylglucosaminyltransferase II catalytic domain. Such catalyticdomains are useful for expressing complex N-glycans in non-mammaliancells. N-acetylglucosaminyltransferase I (GlcNAc-TI; GnTI; EC 2.4.1.101)catalyzes the reactionUDP-N-acetyl-D-glucosamine+3-(alpha-D-mannosyl)-beta-D-mannosyl-R<=>UDP+3-(2-(N-acetyl-beta-D-glucosaminyl)-alpha-D-mannosyl)-beta-D-mannosyl-R,where R represents the remainder of the N-linked oligosaccharide in theglycan acceptor. An N-acetylglucosaminyltransferase I catalytic domainis any portion of an N-acetylglucosaminyltransferase I enzyme that iscapable of catalyzing this reaction. N-acetylglucosaminyltransferase II(GlcNAc-TII; GnTII; EC 2.4.1.143) catalyzes the reactionUDP-N-acetyl-D-glucosamine+6-(alpha-D-mannosyl)-beta-D-mannosyl-R<=>UDP+6-(2-(N-acetyl-beta-D-glucosaminyl)-alpha-D-mannosyl)-beta-D-mannosyl-R,where R represents the remainder of the N-linked oligosaccharide in theglycan acceptor. An N-acetylglucosaminyltransferase II catalytic domainis any portion of an N-acetylglucosaminyltransferase II enzyme that iscapable of catalyzing this reaction. Examples of suitableN-acetylglucosaminyltransferase I catalytic domains and anN-acetylglucosaminyltransferase II catalytic domains can be found inInternational Patent Publication WO2012/069593. TheN-acetylglucosaminyltransferase I catalytic domain andN-acetylglucosaminyltransferase II catalytic domain can be encoded by asingle polynucleotide. In certain embodiments, the single polynucleotideencodes a fusion protein containing the N-acetylglucosaminyltransferaseI catalytic domain and the N-acetylglucosaminyltransferase II catalyticdomain. Alternatively, the N-acetylglucosaminyltransferase I catalyticdomain can be encoded by a first polynucleotide and theN-acetylglucosaminyltransferase II catalytic domain can be encoded by asecond polynucleotide.

In embodiments where, the filamentous fungal cell or Trichoderma fungalcell contains an N-acetylglucosaminyltransferase I catalytic domain andan N-acetylglucosaminyltransferase II catalytic domain, the cell canalso contain a polynucleotide encoding a mannosidase II. Mannosidase IIenzymes are capable of cleaving Man5 structures of GlcNAcMan5 togenerate GlcNAcMan3, and if combined with action of a catalytic domainof GnTII, to generate G0; and further, with action of a catalytic domainof a galactosyltransferase, to generate G1 and G2. In certainembodiments mannosidase II-type enzymes belong to glycoside hydrolasefamily 38 (cazy.org/GH38_all.html). Examples of such enzymes includehuman enzyme AAC50302, D. melanogaster enzyme (Van den Eisen J. M. et al(2001) EMBO J. 20: 3008-3017), those with the 3D structure according toPDB-reference 1HTY, and others referenced with the catalytic domain inPDB. For ER/Golgi expression, the catalytic domain of the mannosidase istypically fused with an N-terminal targeting peptide, for example usingtargeting peptides listed in the International Patent Publication No.WO2012/069593 or of SEQ ID NOs 589-594. After transformation with thecatalytic domain of a mannosidase II-type mannosidase, a straineffectively producing GlcNAc2Man3, GlcNAc1Man3 or G0 is selected.

In certain embodiments that may be combined with the precedingembodiments, the filamentous fungal cell further contains apolynucleotide encoding a UDP-GlcNAc transporter.

In certain embodiments that may be combined with the precedingembodiments, the filamentous fungal cell further contains apolynucleotide encoding a β-1,4-galactosyltransferase. Generally,ρ-1,4-galactosyltransferases belong to the CAZy glycosyltransferasefamily 7 (cazy.org/GT7_all.html). Examples of useful β4GalT enzymesinclude β4GalT1, e.g. bovine Bos taurus enzyme AAA30534.1 (Shaper N. L.et al Proc. Natl. Acad. Sci. U.S.A. 83 (6), 1573-1577 (1986)), humanenzyme (Guo S. et al. Glycobiology 2001, 11:813-20), and Mus musculusenzyme AAA37297 (Shaper, N. L. et al. 1998 J. Biol. Chem. 263 (21),10420-10428). In certain embodiments of the invention where thefilamentous fungal cell contains a polynucleotide encoding agalactosyltransferase, the filamentous fungal cell also contains apolynucleotide encoding a UDP-Gal 4 epimerase and/or UDP-Galtransporter. In certain embodiments of the invention where thefilamentous fungal cell contains a polynucleotide encoding agalactosyltransferase, lactose may be used as the carbon source insteadof glucose when culturing the host cell. The culture medium may bebetween pH 4.5 and 7.0 or between 5.0 and 6.5. In certain embodiments ofthe invention where the filamentous fungal cell contains apolynucleotide encoding a galactosyltransferase and, optionally, apolynucleotide encoding a UDP-Gal 4 epimerase and/or UDP-Galtransporter, a divalent cation such as Mn2+, Ca2+ or Mg2+ may be addedto the cell culture medium.

In certain embodiments that may be combined with the precedingembodiments, the level of activity of alpha-1,6-mannosyltransferase inthe host cell is reduced compared to the level of activity in awild-type host cell. In certain embodiments, the filamentous fungal hasa reduced level of expression of an och1 gene compared to the level ofexpression in a wild-type filamentous fungal cell.

Another aspect includes methods of producing a Man3GlcNAc2 N-glycan[i.e. Manα3(Manα6)Manβ4GlcNAcβ4GlcNAc] in a filamentous fungal cellincluding the steps of providing a filamentous fungal cell with arecombinant polynucleotide encoding a heterologous polypeptide and areduced level of activity of an alg3 mannosyltransferase compared to thelevel of activity in a wild-type filamentous fungal cell and culturingthe filamentous fungal cell to produce a Man3GlcNAc2 glycan, where theMan3GlcNAc2 glycan constitute at least 10%, at least 20%, at least 30%,at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or 100% (mol %) of the neutral N-glycans secreted by thefilamentous fungal cell. In certain embodiment, Man3GlcNAc2 N-glycanrepresents at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or100% (mol %) of the total N-glycans of the heterologous polypeptide.

Another aspect includes methods of producing a complex N-glycan (i.e anN-glycan comprising a terminal GlcNAc2Man3 structure), for exampleGlcNAc2Man3GlcNAc2 {i.e. G0, i.e.GlcNAcβ2Manα3(GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAc} glycan in a filamentousfungal cell including the steps of providing a filamentous fungal cellwith a recombinant polynucleotide encoding a heterologous polypeptide, areduced level of activity of an alg3 mannosyltransferase compared to thelevel of activity in a wild-type filamentous fungal cell and comprisingfurther a polynucleotide encoding an N-acetylglucosaminyltransferase Icatalytic domain and a polynucleotide encoding anN-acetylglucosaminyltransferase II catalytic domain and culturing thefilamentous fungal cell to produce the complex N-glycan, for exampleGlcNAc2Man3GlcNAc2 glycan, where the GlcNAc2Man3GlcNAc2 glycanconstitutes at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or 100% (mol %) of the neutral N-glycans secreted by thefilamentous fungal cell. In certain embodiments, the complex N-glycan,for example GlcNAc2Man3GlcNAc2 glycan, represents at least 5%, at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, or 100% (mol %) of thetotal N-glycans of the polypeptide. In certain embodiments, said complexN-glycans are GlcNAcMan3 and/or GlcNAc2Man3.

Another aspect includes methods of producing a G1 or G2 N-glycan ormixture thereof, for example GalGlcNAc2Man3GlcNAc2 {i.e. G1, i.e.Galβ4GlcNAcβ2Manα3(GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAc} orGlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAc} and/orGal2GlcNAc2Man3GlcNAc2 {i.e. G2, i.e.Galβ4GlcNAcβ2Manα3(Galβ4GlcNAcβ2Manα6)Manβ4GlcNAcβ4GlcNAc} glycan in afilamentous fungal cell including the steps of providing a filamentousfungal cell with a recombinant polynucleotide encoding a heterologouspolypeptide and a reduced level of activity of an alg3mannosyltransferase compared to the level of activity in a wild-typefilamentous fungal cell and comprising further a polynucleotide encodingan N-acetylglucosaminyltransferase I catalytic domain, a polynucleotideencoding an N-acetylglucosaminyltransferase II catalytic domain, and apolynucleotide encoding a GalT catalytic domain and culturing thefilamentous fungal cell to produce the G1 or G2 N-glycan or mixturethereof, where G1 glycan constitutes at least 5%, at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, or 100% (mol %) of the neutralN-glycans secreted by the filamentous fungal cell, or where the G2glycan constitutes at least 5%, at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, or 100% (mol %) of the neutral N-glycans secreted bythe filamentous fungal cell. In certain embodiment, G1 glycanconstitutes at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, or100% (mol %) of the total N-glycans of the polypeptide. In certainembodiment, G2 glycan constitutes at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, or 100% (mol %) of the total N-glycans of thepolypeptide.

In certain embodiments, the method of producing a complex N-glycan willgenerate a mixture of different glycans. The complex N-glycan orMan3GlcNAc2 may constitute at least 5%, at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%), or at least 90% or more of such a glycan mixture. In certainembodiments, at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%), orat least 90% or more of the N-glycans of the polypeptide consists ofsuch a glycan mixture. In certain embodiments, the method of producing acomplex and G1 and/or G2 N-glycan will generate a mixture of differentglycans. The complex N-glycan, Man3GlcNAc2, G1 and/or G2 may constituteat least 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%), or at least 90% ormore of such a glycan mixture. In certain embodiments, at least 5%, atleast 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%), or at least 90% or more of theN-glycans of the polypeptide consists of such a glycan mixture.

In certain embodiments, methods of producing a hybrid N-glycan aredesirable. As used herein, the term “hybrid” means a glycan containingboth unsubstituted terminal mannose residues (as are present inhigh-mannose glycans) and substituted mannose residues with anN-acetylglucosamine linkage, for exampleGlcNAcβ2Manα3[Manα3(Manα6)Manα6]Manβ4GlcNAcβ4GlcNAc. In suchembodiments, a Man5 {i.e Manα3 [Manα3(Manα6) Manα6]Manβ4GlcNAcβ4GlcNAc}expressing filamentous fungal cell such as T. reesei strain istransformed with a recombinant polynucleotide encoding a heterologouspolypeptide and a polynucleotide encoding anN-acetylglucosaminyltransferase I catalytic domain and the filamentousfungal cell is cultured to produce the hybrid N-glycan where the hybridN-glycan constitutes at least 5%, at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, or 100% (mol %) of the neutral N-glycans secreted bythe filamentous fungal cell. In certain embodiment, at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or 100% (mol %) of the N-glycansof the polypeptide consists of a hybrid N-glycan.

The Man3GlcNAc2, complex, hybrid, G1, and G2 N-glycan may be attached toa molecule selected from an amino acid, a peptide, and a polypeptide. Incertain embodiments, the Man3GlcNAc2, complex, hybrid, G1, and G2N-glycan is attached to a heterologous polypeptide. In certainembodiments, the heterologous polypeptide is a glycosylated protein. Incertain embodiment, the glycosylated polypeptide is a mammalianpolypeptide. In certain embodiments, mammalian polypeptide is anantibody or its antigen-binding fragment.

In certain embodiments, glycosyltransferases, for example, GnTI, GnTII,or GalT or glycosylhydrolases, for example, α-1,2-mannosidase ormannosidase II, include a targeting peptide linked to the catalyticdomains. The term “linked” as used herein means that two polymers ofamino acid residues in the case of a polypeptide or two polymers ofnucleotides in the case of a polynucleotide are either coupled directlyadjacent to each other or are within the same polypeptide orpolynucleotide but are separated by intervening amino acid residues ornucleotides. A “targeting peptide”, as used herein, refers to any numberof consecutive amino acid residues of the recombinant protein that arecapable of localizing the recombinant protein to the endoplasmicreticulum (ER) or Golgi apparatus (Golgi) within the filamentous fungalcell. The targeting peptide may be N-terminal or C-terminal to thecatalytic domains. In certain embodiments, the targeting peptide isN-terminal to the catalytic domains. In certain embodiments, thetargeting peptide provides direct binding to the ER or Golgi membrane.Components of the targeting peptide may come from any enzyme thatnormally resides in the ER or Golgi apparatus. Such enzymes includemannosidases, mannosyltransferases, glycosyltransferases, Type 2 Golgiproteins, and MNN2, MNN4, MNN6, MNN9, MNN10, MNS1, KRE2, VAN1, and OCH1enzymes. Suitable targeting peptides are described in the InternationalPatent Publication No. WO2012/069593. In one embodiment, the targetingpeptide of GnTI or GnTII is human GnTII enzyme. In other embodiments,targeting peptide is derived from Trichoderma Kre2, Kre2-like, Och1,Anp1, and Van1. In one embodiment, the targeting peptide is selectedfrom the group of SEQ ID NOs: 589-594.

Uses of the Filamentous Fungal Cells of the Invention

The invention herein further relates to methods of using any of thefilamentous fungal cells of the present disclosure, such as Trichodermafungal cells, that have reduced or no protease activity of at leastthree proteases and that contain a recombinant polynucleotide encoding aheterologous polypeptide, such as a mammalian polypeptide, that isproduced at increased levels, for improving heterologous polypeptidestability and for making a heterologous polypeptide. Methods ofmeasuring protein stability and for making a heterologous polypeptideare well known, and include, without limitation, all the methods andtechniques described in the present disclosure.

Accordingly, certain embodiments of the present disclosure relate tomethods of improving heterologous polypeptide stability, by: a)providing a filamentous fungal cell of the present disclosure havingreduced or no activity of at least three proteases, where the cellfurther contains a recombinant polynucleotide encoding a heterologouspolypeptide; and b) culturing the cell such that the heterologouspolypeptide is expressed, where the heterologous polypeptide hasincreased stability compared to a host cell not containing the mutationsof the genes encoding the proteases. Other embodiments of the presentdisclosure relate to methods of improving mammalian polypeptidestability, by: a) providing a Trichoderma fungal cell of the presentdisclosure having reduced or no activity of at least three proteases,where the cell further contains a recombinant polynucleotide encoding amammalian polypeptide; and b) culturing the cell such that the mammalianpolypeptide is expressed, where the mammalian polypeptide has increasedstability compared to a host cell not containing the mutations of thegenes encoding the proteases. The filamentous fungal cell or Trichodermafungal cell may be any cell described in the section entitled“Filamentous Fungal Cells of the Invention”. Methods of measuringpolypeptide stability and for culturing filamentous fungal andTrichoderma fungal cells are well known in the art, and include, withoutlimitation, all the methods and techniques described in the presentdisclosure.

In certain embodiments, the stability of the heterologous polypeptide ormammalian polypeptide is increased by at least 2-fold, at least 3-fold,at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, atleast 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, atleast 20-fold, at least 25-fold, at least 30-fold, at least 40-fold, atleast 50-fold, at least 60-fold, at least 70-fold, at least 75-fold, atleast 80-fold, at least 90-fold, at least 100-fold, or a greater foldhigher compared to a heterologous polypeptide or mammalian polypeptideexpressed in a corresponding parental filamentous fungal or Trichodermafungal cell.

Other embodiments of the present disclosure relate to methods of makinga heterologous polypeptide, by: a) providing a filamentous fungal cellof the present disclosure having reduced or no activity of at leastthree proteases, where the cell further contains a recombinantpolynucleotide encoding a heterologous polypeptide; b) culturing thehost cell such that the heterologous polypeptide is expressed; and c)purifying the heterologous polypeptide. Further embodiments of thepresent disclosure relate to methods of making a mammalian polypeptide,by: a) providing a Trichoderma fungal cell of the present disclosurehaving reduced or no activity of at least three protease, where the cellfurther contains a recombinant polynucleotide encoding a mammalianpolypeptide; b) culturing the host cell such that the mammalianpolypeptide is expressed; and c) purifying the mammalian polypeptide.The filamentous fungal cell or Trichoderma fungal cell may be any celldescribed in the section entitled “Filamentous Fungal Cells of theInvention”. Methods of culturing filamentous fungal and Trichodermafungal cells and purifying polypeptides are well known in the art, andinclude, without limitation, all the methods and techniques described inthe present disclosure.

In certain embodiments, the filamentous fungal cell or Trichodermafungal cell is cultured at a pH range selected from pH 3.5 to 7; pH 3.5to 6.5; pH 4 to 6; pH 4.3 to 5.7; pH 4.4 to 5.6; and pH 4.5 to 5.5. Incertain embodiments, to produce an antibody the filamentous fungal cellor Trichoderma fungal cell is cultured at a pH range selected from 4.7to 6.5; pH 4.8 to 6.0; pH 4.9 to 5.9; and pH 5.0 to 5.8.

In some embodiments, the heterologous polypeptide is a mammalianpolypeptide. In other embodiments, the heterologous polypeptide is anon-mammalian polypeptide.

In certain embodiments, the mammalian polypeptide is selected from animmunoglobulin, immunoglobulin heavy chain, an immunoglobulin lightchain, a monoclonal antibody, a hybrid antibody, an F(ab′)2 antibodyfragment, an F(ab) antibody fragment, an Fv molecule, a single-chain Fvantibody, a dimeric antibody fragment, a trimeric antibody fragment, afunctional antibody fragment, a single domain antibody, multimericsingle domain antibodies, an immunoadhesin, insulin-like growth factor1, a growth hormone, insulin, and erythropoietin. In other embodiments,the mammalian protein is an immunoglobulin or insulin-like growthfactor 1. In yet other embodiments, the mammalian protein is anantibody. In further embodiments, the yield of the mammalian polypeptideis at least 0.5, at least 1, at least 2, at least 3, at least 4, or atleast 5 grams per liter. In certain embodiments, the mammalianpolypeptide is an antibody, optionally, IgG1, IgG2, IgG3, or IgG4. Infurther embodiments, the yield of the antibody is at least 0.5, at least1, at least 2, at least 3, at least 4, or at least 5 grams per liter. Instill other embodiments, the mammalian polypeptide is a growth factor ora cytokine. In further embodiments, the yield of the growth factor orcytokine is at least 0.1, at least 0.2, at least 0.3, at least 0.4, atleast 0.5, at least 1, at least 1.5, at least 2, at least 3, at least 4,or at least 5 grams per liter. In further embodiments, the mammalianpolypeptide is an antibody, and the antibody contains at least 70%, atleast 80%, at least 90%, at least 95%, or at least 98% of a naturalantibody C-terminus and N-terminus without additional amino acidresidues. In other embodiments, the mammalian polypeptide is anantibody, and the antibody contains at least 70%, at least 80%, at least90%, at least 95%, or at least 98% of a natural antibody C-terminus andN-terminus that do not lack any C-terminal or N-terminal amino acidresidues

In certain embodiments where the mammalian polypeptide is purified fromcell culture, the culture containing the mammalian polypeptide containspolypeptide fragments that make up a mass percentage that is less than50%, less than 40%, less than 30%, less than 20%, or less than 10% ofthe mass of the produced polypeptides. In certain preferred embodiments,the mammalian polypeptide is an antibody, and the polypeptide fragmentsare heavy chain fragments and/or light chain fragments. In otherembodiments, where the mammalian polypeptide is an antibody and theantibody purified from cell culture, the culture containing the antibodycontains free heavy chains and/or free light chains that make up a masspercentage that is less than 50%, less than 40%, less than 30%, lessthan 20%, or less than 10% of the mass of the produced antibody. Methodsof determining the mass percentage of polypeptide fragments are wellknown in the art and include, measuring signal intensity from anSDS-gel.

In further embodiments, the non-mammalian polypeptide is selected froman aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase,cellulase. chitinase, cutinase, deoxyribonuclease, esterase,alpha-galactosidase, beta-galactosidase, glucoamylase,alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase,mutanase, oxidase, pectinolytic enzyme, peroxidase, phospholipase,phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease,transglutaminase, and xylanase.

In certain embodiments of any of the disclosed methods, the methodincludes the further step of providing one or more, two or more, threeor more, four or more, or five or more protease inhibitors. In certainembodiments, the protease inhibitors are peptides that are co-expressedwith the mammalian polypeptide. In other embodiments, the inhibitorsinhibit at least two, at least three, or at least four proteases from aprotease family selected from aspartic proteases, trypsin-like serineproteases, subtilisin proteases, and glutamic proteases.

In certain embodiments of any of the disclosed methods, the filamentousfungal cell or Trichoderma fungal cell also contains a carrier protein.As used herein, a “carrier protein” is portion of a protein that isendogenous to and highly secreted by a filamentous fungal cell orTrichoderma fungal cell. Suitable carrier proteins include, withoutlimitation, those of T. reesei mannanase I (Man5A, or MANI), T. reeseicellobiohydrolase II (Cel6A, or CBHII) (see, e.g., Paloheimo et al Appl.Environ. Microbiol. 2003 December; 69(12): 7073-7082) or T. reeseicellobiohydrolase I (CBHI). In some embodiments, the carrier protein isCBH1. In other embodiments, the carrier protein is a truncated T. reeseiCBH1 protein that includes the CBH1 core region and part of the CBH1linker region. In some embodiments, a carrier such as acellobiohydrolase or its fragment is fused to an antibody light chainand/or an antibody heavy chain. In some embodiments, a carrier such as acellobiohydrolase or its fragment is fused to insulin-like growth factor1, growth hormone, insulin, interferon alpha 2b, fibroblast growthfactor 21, or human serum albumin. In some embodiments, acarrier-antibody fusion polypeptide comprises a Kex2 cleavage site. Incertain embodiments, Kex2, or other carrier cleaving enzyme, isendogenous to a filamentous fungal cell. In certain embodiments, carriercleaving protease is heterologous to the filamentous fungal cell, forexample, another Kex2 protein derived from yeast or a TEV protease. Incertain embodiments, carrier cleaving enzyme is overexpressed. Incertain embodiments, the carrier consists of about 469 to 478 aminoacids of N-terminal part of the T. reesei CBH1 protein GenBank accessionNo. EGR44817.1. In one embodiment, the polynucleotide encoding theheterologous glycoprotein (e.g. the antibody) further comprises apolynucleotide encoding CBH1 catalytic domain and linker as a carrierprotein, and/or cbh1 promoter. In certain embodiments, the filamentousfungal cell of the invention overexpress KEX2 protease. In an embodimentthe heterologous glycoprotein (e.g. the antibody) is expressed as fusionconstruct comprising an endogenous fungal polypeptide, a protease sitesuch as a Kex2 cleavage site, and the heterologous protein such as anantibody heavy and/or light chain. Useful 2-7 amino acids combinationspreceding Kex2 cleavage site have been described, for example, inMikosch et al. (1996) J. Biotechnol. 52:97-106; Goller et al. (1998)Appl Environ Microbiol. 64:3202-3208; Spencer et al. (1998) Eur. J.Biochem. 258:107-112; Jalving et al. (2000) Appl. Environ. Microbiol.66:363-368; Ward et al. (2004) Appl. Environ. Microbiol. 70:2567-2576;Ahn et al. (2004) Appl. Microbiol. Biotechnol. 64:833-839; Paloheimo etal. (2007) Appl Environ Microbiol. 73:3215-3224; Paloheimo et al. (2003)Appl Environ Microbiol. 69:7073-7082; and Margolles-Clark et al. (1996)Eur J Biochem. 237:553-560.

It is to be understood that, while the invention has been described inconjunction with the certain specific embodiments thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention. Other aspects, advantages, and modifications within the scopeof the invention will be apparent to those skilled in the art to whichthe invention pertains.

The invention having been described, the following examples are offeredto illustrate the subject invention by way of illustration, not by wayof limitation.

EXAMPLES

The identification of certain proteases in Trichoderma reesei andcorresponding T. reesei cells with deficient protease activity have beendescribed in Examples 1-23 of WO 2013/102674 which contents isincorporated herein by reference.

Example 1 Generation of Deletion Plasmids

The deletion plasmid below were constructed essentially as described forpep1 deletion plasmid pTTv41 in Example 1 of WO2013/102674, except thatthe marker used for selection was pyr4-hgh or pyr4 from pTTv213 orpTTv194.

Deletion Plasmid for amp1

958 bp of 5′ flanking region and 994 bp of 3′ flanking region wereselected as the basis of the amp1 deletion plasmid pTTv240. A 367 bpstretch from the end of amp1 5′ flank was used as the direct repeatfragment. These fragments were amplified by PCR using the primers listedin Table 1-1. Template used in the PCR of the flanking regions was fromthe T. reesei wild type strain QM6a. The products were separated withagarose gel electrophoresis and the correct fragments were isolated fromthe gel with a gel extraction kit (Qiagen) using standard laboratorymethods. The pyr4-hph selection marker used in pTTv240 was obtained frompTTv213 (Δpep2-pyr-hph see Example 14 of WO2013/102674) with NotIdigestion. To enable removal of the pyr4-hgh marker cassette, NotIrestriction sites were introduced on both sides of the cassette. AscIsite was introduced between the amp1 5′direct repeat and 3′ flank.Vector backbone was EcoRI/XhoI digested pRS426 and the plasmid pTTv240was constructed with the 5′ flank, 3′ flank, the 5′ direct repeat,pyr4-hph marker, and vector backbone using the yeast homologousrecombination method as described in Example 1 of WO2013/102674). Thedeletion plasmid for amp1 (pTTv240, Table 1-1) results in a deletion inthe amp1 locus and covers the complete coding sequence of AMP1(tre81070).

TABLE 1-1 Primers for generating amp1 deletion plasmid.Deletion plasmid pTTv240 (Δamp1-pyr4-hph), vector backbone pRS426 PrimerSequence T832_amp1_5flkfw_ GTAACGCCAGGGTTTTCCCAGTCACGACGGTTTAAACC vectorATGGAAGATGCGAGCTACA (SEQ ID NO: 806) T833_amp1_5flkrev_GCGCTGGCAACGAGAGCAGAGCAGCAGTAGTCGATGC pyr4PromTAGGCGGCCGCGGAGAGGAGATGGGTGTTGA  (SEQ ID NO: 807) T836_amp1_3flkfw_CCCCCCTTTCTCTCTCTCTTTCAACACCCATCTCCTCTC 5DR_endCGGCGCGCCGCGAGGTGCGTTTCTGTAGC  (SEQ ID NO: 808) T837_amp1_3flkrev_GCGGATAACAATTTCACACAGGAAACAGCGTTTAAAC vectorCGGCAAATACTACGACGACA (SEQ ID NO: 809) T834_amp1_5 DR fwdGTACACTTGTTTAGAGGTAATCCTTCTTTCTAGAAGGA GAGCGGCCGCGTCGAGTGCATCAATGACGA (SEQ ID NO: 810) T835_amp1_5 DR revCAAACAGCATGCTCGTAAATGCTACAGAAACGCACCT CGCGGCGCGCCGGAGAGGAGATGGGTGTTGA (SEQ ID NO: 811)Deletion Plasmid for amp2

918 bp of 5′ flanking region and 978 bp of 3′ flanking region wereselected as the basis of the amp2 deletion plasmid pTTv271. Thesefragments were amplified by PCR using the primers listed in Table 1-2.Template used in the PCR of the flanking regions was from the T. reeseiwild type strain QM6a. The products were separated with agarose gelelectrophoresis and the correct fragments were isolated from the gelwith a gel extraction kit (Qiagen) using standard laboratory methods.The pyr4-hph cassette was obtained from pTTv194 (Δpep4-pyr-hph) withNotI digestion. To enable removal of the marker cassette, NotIrestriction sites were introduced on both sides of the cassette. Vectorbackbone was EcoRI/XhoI digested pRS426 and the plasmid pTTv271 wasconstructed with the 5′ flank, 3′ flank, pyr4-hph marker, and vectorbackbone using the yeast homologous recombination method.

Another deletion plasmid for amp2 (pTTv327) was generated as follows. A311 bp stretch from the end of amp2 5′ flank was used as the directrepeat fragment. The fragments were amplified by PCR using the primerslisted in Table 1-3. The products were separated with agarose gelelectrophoresis and the correct fragments were isolated from the gel.The pyr4-hph cassette was obtained from pTTv210 (Δsep1-pyr4-hph) withNotI digestion. To enable removal of the marker cassette, NotIrestriction sites were introduced on both sides of the cassette. AscIsite was introduced between the amp2 5′direct repeat and 3′ flank.Vector backbone was EcoRI/XhoI digested pRS426 and the plasmid wasconstructed as described in Example 1 of WO2013/102674.

These deletion plasmids for amp2 (pTTv271 and pTTv327) result in 2143 bpdeletion in the amp2 locus and cover the complete coding sequence ofAMP2 (tre108592).

TABLE 1-2 Primers for generating amp2 deletion plasmid.Deletion plasmid pTTv271 (Δamp2-pyr4-hph), vector backbone pRS426 PrimerSequence T1079_amp2_5flkfw_ GTAACGCCAGGGTTTTCCCAGTCACGACGGTTTAAACCCvector ATTCTCGTCGTTGTTTCC (SEQ ID NO: 812) T1080_amp2_5flkrev_GCGCTGGCAACGAGAGCAGAGCAGCAGTAGTCGATGCTAGGC pyr4PromGGCCGCTGGAGGAGTAGCTGCACTGA (SEQ ID NO: 813) T1081_amp2_3flkfw_CAACCAGCCGCAGCCTCAGCCTCTCTCAGCCTCATCAGCCGCG pyr4loopGCCGCACAGCCAGTGGAAACCAAAC (SEQ ID NO: 814) T1082_amp2_3flkrev_GCGGATAACAATTTCACACAGGAAACAGCGTTTAAACTAGAGC vectorTTGGAGGGAACAGG (SEQ ID NO: 815)

TABLE 1-3 Primers for generating amp2 deletion plasmid.Deletion plasmid pTTv327 (Δamp2-pyr4-hph), vector backbone pRS426 PrimerSequence T1079_amp2_5flkfw_ GTAACGCCAGGGTTTTCCCAGTCACGACGGTTTAAAC vecforCCATTCTCGTCGTTGTTTCC (SEQ ID NO: 812) T1080_amp2_5flkrev_GCGCTGGCAACGAGAGCAGAGCAGCAGTAGTCGAT pyr4PromGCTAGGCGGCCGCTGGAGGAGTAGCTGCACTGA  (SEQ ID NO: 813) T1194_amp2_5dr_fGTACACTTGTTTAGAGGTAATCCTTCTTTCTAGAAGGAGAGCGGCCGCGAGTCGGTCCTACTGCTTGA (SEQ ID NO: 816) T1195_amp2_5dr_rTTTTACTTGTTTTGATAAGGGTTTGGTTTCCACTGGCTGTGGCGCGCCTGGAGGAGTAGCTGCACTGA (SEQ ID NO: 817) T1196_amp2_3f_f_2ACAGCCAGTGGAAACCAAAC (SEQ ID NO: 818) T1082_amp2_3flkrev_GCGGATAACAATTTCACACAGGAAACAGCGTTTAAAC vectorTAGAGCTTGGAGGGAACAGG (SEQ ID NO: 815)Deletion Plasmid for sep1

First deletion plasmid for sep1, pTTv210, contained 1099 bp 5′ flankingregion and 806 bp 3′ flanking region, a 300 bp strech from the end ofsep1 5′ flank as the direct repeat fragment, pyr4-hph double selectionmarker and an expression construct for T. reesei native kex2 (tre123561;promoter cDNA1-kex2-terminator cbh2). All other fragments except pyr4were amplified by PCR using the primers listed in Table 1-4. Templateused in the PCR of the flanking regions, direct repeat, kex2 andterminator was from the T. reesei wild type strain QM6a. Templates forcDNA1 promoter and hph marker were plasmids. The pyr4 marker wasobtained from pTTv181 with NotI digestion (pTTv181 is described inExample 4 of WO2013/102674). To enable removal of the pyr4-hph markercassette, NotI restriction sites were introduced on both sides of thecassette. AscI site was introduced between the sep1 5′direct repeat and3′ flank. The products were separated with agarose gel electrophoresisand the correct fragments were isolated from the gel with a gelextraction kit (Qiagen). Vector backbone was EcoRI/XhoI digested pRS426.Plasmid pTTv210 was constructed with all eight fragments above andvector backbone using the yeast homologous recombination method asdescribed above.

The second deletion plasmid for sep1, pTTv247, was constructed byremoving the KEX2 overexpression cassette from pTTv210 with AscIdigestion. The fragments were separated with agarose gel electrophoresisand the vector part isolated from the gel with a gel extraction kit(Qiagen). pTTv247 was cloned by self-ligation using T4 DNA ligase andstandard laboratory methods.

Third deletion plasmid pTTv255 for sep1 was constructed as follows. 1099bp of 5′ flanking region and 806 bp of 3′ flanking region were selectedas the basis and a 300 bp stretch from the end of sep1 5′ flank was usedas the direct repeat fragment. These fragments were amplified by PCRusing the primers listed in Table 1-4. The pyr4 cassette was obtainedfrom pTTv181 (Δpep4-pyr4) with NotI digestion. To enable removal of themarker cassette, NotI restriction sites were introduced on both sides ofthe cassette. AscI site was introduced between the sep1 5′direct repeatand 3′ flank. Vector backbone was EcoRI/XhoI digested pRS426 and theplasmid was constructed using the yeast homologous recombination asabove.

The deletion plasmids pTTv210, pTTv247 and pTTv255 result in 2519 bpdeletion in the sep1 locus and cover the complete coding sequence ofSEP1 (tre124051).

TABLE 1-4 Primers for generating sep1 deletion plasmids. PrimerPrimer sequence Deletion plasmid pTTv210 (Δsep1-pyr4-hph,kex2), vector backbone pRS426 T489_serendo_5f_for GGTAACGCCAGGGTTTTCCCAGTCACGACGGTTTAAAC ATGGGCTGAAACCGGCGCA A (SEQ ID NO: 819)T490_serendo_5f_rev GCGCTGGCAACGAGAGCAG AGCAGCAGTAGTCGATGCTAGGCGGCCGCGACAGCGCC TCGCCAAGTGT (SEQ ID NO: 820) T493_serendo_DR_forGTACACTTGTTTAGAGGTA ATCCTTCTTTCTAGAAGGA GAGCGGCCGCAGCAGCCTG CCCAGAGAATC(SEQ ID NO: 821) T494_serendo_DR_rev GTCATTAAGTCCATCATTCCACGTCCTTCAGACCGAAT TCGGCGCGCCGACAGCGCC TCGCCAAGTGT (SEQ ID NO: 822)T498_serendo_3f_for ATGATGCCTTTGCAGAAAT GGCTTGCTCGCTGACTGATACGGCGCGCCTGGCGCTTC CGTTCCCTTCC (SEQ ID NO: 823) T499_serendo_3f_revAGCGGATAACAATTTCACA CAGGAAACAGCGTTTAAAC TGTTGAGACGGGCGAGTGCT (SEQ ID NO: 824) T491_hph_recpyr4_for3 TGATTGTACCCCAGCTGCGATTGATGTGTATCTTTGCA TGATTTAAATTCTCCTTAG CTCTGTACAGT (SEQ ID NO: 607)T492_hph_rev2 GCGGCCGCTCTCCTTCTAG AAAGAAGGA (SEQ ID NO :608)T495_cDNA1_for GAATTCGGTCTGAAGGACG T (SEQ ID NO: 609) T138_cDNA1_RevGTTGAGAGAAGTTGTTGGA TTG (SEQ ID NO: 392) T139_123561For_cDNA1AACCAAAGACTTTTTGATC AATCCAACAACTTCTCTCA ACATGAAGATTTCCTCGAT CCTTG(SEQ ID NO: 393) T516_123561Rev TCAGCGCCGTAACCTCTGC (SEQ ID NO: 394)T496_tcbh2_for TGATGGTGATGAGGCGGAA AAGCAGAGGTTACGGCGCTGAGGCTTTCGTGACCGGGC TTC (SEQ ID NO: 610) T497_tcbh2_revGTATCAGTCAGCGAGCAAG C (SEQ ID NO: 611)Deletion plasmid pTTv247 (Δsep1-pyr4-hph), vector backbone pTTv210No new primers; pTTv210 digested with AscI (to remove kex2 cassette)and ~12 kb fragment self-ligated Deletion plasmid pTTv255 (Δsep1-pyr4),vector backbone pRS426 T489_serendo_5f_for GGTAACGCCAGGGTTTTCCCAGTCACGACGGTTTAAACAT GGGCTGAAACCGGCGCAA (SEQ ID NO: 819)T490_serendo_5f_rev GCGCTGGCAACGAGAGCAGA GCAGCAGTAGTCGATGCTAGGCGGCCGCGACAGCGCCTCG CCAAGTGT (SEQ ID NO: 820) T1000_serendo_5dr_for2TGATTGTACCCCAGCTGCGA TTGATGTGTATCTTTGCATG GCGGCCGCAGCAGCCTGCCC AGAGAATC(SEQ ID NO: 825) T1001_serendo_5dr_rev2 GACAATCAGAGGCCTCAATTGGAAGGGAACGGAAGCGCCA GGCGCGCCGACAGCGCCTCG CCAAGTGT (SEQ ID NO: 826)T502_serendo_3f_probef TGGCGCTTCCGTTCCCTTCC (SEQ ID NO: 827)T499_serendo_3f_rev AGCGGATAACAATTTCACAC AGGAAACAGCGTTTAAACTGTTGAGACGGGCGAGTGCT (SEQ ID NO: 824)Deletion Plasmids for pep9

918 bp of 5′ flanking region and 978 bp of 3′ flanking region wereselected as the basis of the pep9 deletion plasmid pTTv267. Thesefragments were amplified by PCR using the primers listed in Table 1-5.Template used in the PCR of the flanking regions was from the T. reeseiwild type strain QM6a. The products were separated with agarose gelelectrophoresis and the correct fragments were isolated from the gelwith a gel extraction kit (Qiagen). The pyr4-hph cassette was obtainedfrom pTTv194 (Δpep4-pyr-hph) with NotI digestion. To enable removal ofthe marker cassette, NotI restriction sites were introduced on bothsides of the cassette. Vector backbone was EcoRI/XhoI digested pRS426and the plasmid was constructed with the 5′ flank, 3′ flank, pyr4-hphmarker, and vector backbone using the yeast homologous recombinationmethod. This deletion plasmid for pep9 (pTTv267, Table 1-5) results in a1448 bp deletion in the pep9 locus and covers majority of the codingsequence PEP9 (tre79807).

TABLE 1-5 Primers for generating pep9 deletion plasmid.Deletion plasmid pTTv267 (Δpep9-pyr4-hph), vector backbone pRS426 PrimerSequence T1027_pep9_5flkfw_vector GTAACGCCAGGGTTTTCCCAGTCACGACGGTTTAAACA ACCACGACGAAAATCAAGG (SEQ ID NO: 828)T1028_pep9_5flkrev_pyr4Prom GCGCTGGCAACGAGAGCAG AGCAGCAGTAGTCGATGCTAGGCGGCCGCAATGGACCC AGATGTCAAGG (SEQ ID NO: 829)T1029_pep9_3flkfw_pyr4loop CAACCAGCCGCAGCCTCAG CCTCTCTCAGCCTCATCAGCCGCGGCCGCGATCTAGGA TTCGCCAAACG (SEQ ID NO: 830)T1030_pep9_3flkrev_vector GCGGATAACAATTTCACAC AGGAAACAGCGTTTAAACACGACATGAACAAACGGACA (SEQ ID NO: 831)

The second deletion plasmid for pep9, pTTv421, was constructed using theplasmid pTTv267 above as the backbone. The pyr4-hph double marker withpyr4 5DR was removed from pTTv267 with NotI digestion. New marker,pyr4-hph, was obtained from a plasmid derived from pTTv264 with egl2deletion with NotI digestion. To enable removal of the marker cassette,NotI restriction sites were introduced on both sides of the cassette.FseI site was introduced between the pep9 3′direct repeat and 5′ flank A321 bp stretch from the beginning of pep9 3′ flank was used as thedirect repeat fragment. Part of the cbh2 terminator was used as a bridgein cloning. These two fragments were amplified by PCR using the primerslisted in Table 1-6.

The third deletion plasmid for pep9, pTTv426, was constructed using theplasmid pTTv421 above as the backbone. The pyr4-hph double marker wasremoved from pTTv421 with NotI digestion. The pyr4 marker gene wasobtained from pTTv181 (Δpep4-pyr4) with NotI digestion. Cloning of theplasmid pTTv426 was done with quick ligation using T4 DNA ligase at roomtemperature. Part of the ligation mixture was transformed into E. coliwith electroporation. A few clones were cultivated, plasmid DNA wasisolated and digested to screen for correct ligation using standardlaboratory methods. Correct ligation and orientation of the marker wasfurther verified by sequencing. These deletion plasmids for pep9(pTTv421 and pTTv426, Table 1-6) result in a 1448 bp deletion in thepep9 locus and cover majority of the coding sequence of PEP9 (tre79807).

TABLE 1-6 Primers for generating pep9 deletionplasmids pTTv421 and pTTv426. Primer Primer sequenceDeletion plasmid pTTv421 (Δpep9-pyr4-hph), vector backbone pTTv267T1520_pep9_3dr_for TAATCTCTAATCACCTAATA CCTTGACATCTGGGTCCATTGGCCGGCCGATCTAGGATTC GCCAAACG (SEQ ID NO: 832) T1521_pep9_3dr_revGCGCTGGCAACGAGAGCAGA GCAGCAGTAGTCGATGCTAG GCGGCCGCAGTATCGCAGGA GCGAGCAT(SEQ ID NO: 833) T1404_cbh2term_for CCGTCTAGCGCTGTTGATTG(SEQ ID NO: 834) T1522_tcbh2_recpep9_rev GCTGGATGAACGTTTGGCGAATCCTAGATCGCGGCCGCGT GCTGCGGAATCATTATCA (SEQ ID NO: 835)Deletion plasmid pTTv426 (Δpep9-pyr4), vector backbone pTTv421No new primers; pTTv421 digested with NotIand ligated with pyr4 from pTTv181.Deletion Plasmid for slp7

The deletion plasmid pTTv329 for slp7 (tre123865) was constructedessentially as described for pTTv269 except the additional primers usedin Table 1-7, the pyr4-hph cassette was obtained from pTTv210(Δsep1-pyr4-hph), and AscI site was introduced between the slp7 5′directrepeat and 3′ flank.

TABLE 1-7 Additional primers for generatingslp7 deletion plasmid pTTv329.Deletion plasmid pTTv329 (Δslp7-pyr4-hph), vector backbone pRS426 PrimerPrimer sequence T1200_slp7_5dr_f GTACACTTGTTTAGAGGTAATCCTTCTTTCTAGAAGGAGAGCGGCCGCGGGGAAACAA TGACATACGC (SEQ ID NO: 836)T1201_slp7_5dr_r CTCTTGGAGAGTCTTCTCCAAAACCCAAGCTTATCACCCAGGCGCGCCTTTGCAGCAA GATGTCGTTC (SEQ ID NO: 837)T1202_slp7_3f_f2 TGGGTGATAAGCTTGGGTTT (SEQ ID NO: 838)Deletion Plasmid for slp3

The deletion plasmid pTTv116 for slp3 (tre123234) is described inExample 3 of WO2013/102674. This plasmid results in 1597 bp deletion inthe slp3 locus and covers the complete coding sequence of SLP3.

Second deletion plasmid for slp3, pTTv420, was cloned using pTTv116 asbackbone. Marker from pTTv116 was removed by NotI digestion. New marker,pyr4-hph, was obtained from a plasmid derived from pTTv264 with egl2deletion with NotI digestion. To enable removal of the marker cassette,NotI restriction sites were introduced on both sides of the cassette.FseI site was introduced between the slp3 3′direct repeat and 5′ flank.A 300 bp stretch from the beginning of slp3 3′ flank was used as thedirect repeat fragment. Part of the cbh2 terminator was used as a bridgein cloning. These two fragments were amplified by PCR using the primerslisted in Table 1-8. The products were separated with agarose gelelectrophoresis and the correct fragments were isolated from the gelwith a gel extraction kit (Qiagen). The plasmid was constructed usingthe yeast homologous recombination method as described above.

The third deletion plasmid for slp3, pTTv425, was constructed using theplasmid pTTv420 above as the backbone. The pyr4-hph double marker wasremoved from pTTv420 with NotI digestion. The pyr4 marker gene wasobtained from pTTv181 (Δpep4-pyr4) with NotI digestion. Cloning of theplasmid pTTv425 was done with quick ligation using T4 DNA ligase at roomtemperature. Part of the ligation mixture was transformed into E. coliwith electroporation. A few clones were cultivated, plasmid DNA wasisolated and digested to screen for correct ligation using standardlaboratory methods. Correct ligation and orientation of the marker wasfurther verified by sequencing. These three deletion plasmids for slp3(pTTv116, pTTv420 and pTTv425; Table 1-8) result in 1597 bp deletion inthe slp3 locus and cover the complete coding sequence of SLP3.

TABLE 1-8 Primers for generating slp3deletion plasmids pTTv420 and pTTv425. Primer Primer sequenceDeletion plasmid pTTv420 (Δslp3-pyr4-hph), vector backbone pTTv116T1523_slp3_3dr_for TCTTCTTGACACAATG TTCCTGTTCTCCTCAT CCTTGGATGGCCGGCCACCTAATGGTTTCTTC GTTT (SEQ ID NO: 839) T1524_slp3_3dr_revGCGCTGGCAACGAGAG CAGAGCAGCAGTAGTC GATGCTAGGCGGCCGC CTGGAGAGGATATGGT TCTG(SEQ ID NO: 840) T1404_cbh2term_for CCGTCTAGCGCTGTTG ATTG(SEQ ID NO: 834) T1525_tcbh2_recslp3_rev CACCAAAGAAAAACGAAGAAACCATTAGGTGC GGCCGCGTGCTGCGGA ATCATTATCA (SEQ ID NO: 841)Deletion plasmid pTTv425 (Δslp3-pyr4), vector backbone pTTv420No new primers; pTTv420 digested with NotI and ligated with pyr4 from pTTv181.Deletion Plasmids for pep2 with slp2 RNAi

The selection markers in vectors pTTv217 and pTTv263 (both vectorssilencing slp2) were switched to a pyr4-hgh double marker. The vectorswere digested with NotI restriction enzyme and the vector was purifiedfrom agarose gel. The pyr4-hgh cassette was obtained from pTTv194(Δpep4-pyr-hgh) with NotI digestion. The new double marker and thevectors were ligated together and transformed to DH5α chemicallycompetent E. coli. The resulting vectors pTTv376 and pTTv405 wereamplified and purified.

Example 2 Generation of 10-Fold Protease Deletion Strain with sep1Deletion (M659)

To generate a 10-fold protease deletion strain, removal of the pyr4marker was applied to the 9-fold deletion strain M574 (described inExample 14 of WO2013/102674) essentially as described in Example 3 ofWO2013/102674 for removal of the pyr4 blaster cassette from the strainM195 (Δpep1). Consecutive 5-FOA selection steps were carried out toensure that the clones selected were originating from single cells.

Final clones were verified by PCR using the primers listed in Table25-1. Signal corresponding to successful removal of the blaster cassettewas obtained for majority of the clones. Removal of the blaster cassettewas further verified by plating the clones onto minimal medium plateswith or without 5 mM uridine. Resulting strain used in generation of10-fold protease deletion strain was designated with strain number M597.

To remove vector sequence, plasmid pTTv255 (Δpep1-pyr4) was digestedwith MssI and the correct fragment purified from an agarose gel using aQIAquick Gel Extraction Kit (Qiagen). Approximately 5 μg of the deletioncassette was used to transform 9-fold protease deletion strain M597(Δpep1Δtsp1Δslp1Δgap1Δgap2Δpep4Δpep3Δpep5Δpep2, pyr4−). Preparation ofprotoplasts and transformation were carried out essentially as describedfor the strains M181 and M195 using pyr4 selection in WO2013/102674.

Transformants were picked as first streaks. Growing streaks werescreened by PCR (using the primers listed in Table 2-1) for correctintegration. Clones giving the expected signals were purified to singlecell clones and rescreened by PCR using the primers listed in Table 2-1.Deletion of sep1 was verified by Southern analyses from selected clones(FIG. 1 A, B, C) using methods described above. Clone 48-70C wasdesignated with strain number M633. An additional single cellpurification step was applied to strain M633 to obtain 10-fold proteasedeletion strain M659 (clone 48-70C-2).

TABLE 2-1 Primers for screening removal of pyr4blaster cassette from 9-fold protease deletionstrain and for screening pTTv255/Δsep1-pyr4integration and strain purity. Primer SequenceFor screening removal of pyr4 blastercassette from M574 and strain purity T1162_pep2_5f_f2 CTGTAAAGGCAGCATCGG(SEQ ID NO: 638) T1163_pep2_3f_r2 TCAGAACGGCTTCAATCATT (SEQ ID NO: 639)T488_pyr4_5utr_rev GGAGTTGCTTTAATGTCGGG (SEQ ID NO: 428) T601_pep2 fwdGACGTGGTACGACAACATCG (SEQ ID NO: 269) T623_pep2 rev TATCAAGGTACCGGGGACAG(SEQ ID NO: 270) For screening integration of pTTv255 (Δsep1-pyr4)T519_serendo_5int AACCACCTTGTTCTGTCCGT (SEQ ID NO: 842)T488_pyr4_5utr_rev GGAGTTGCTTTAATGTCGGG (SEQ ID NO: 428)T061_pyr4_orf_screen_2F TTAGGCGACCTCTTTTTCCA (SEQ ID NO: 382)T521_serendo_3int GGAACTGTCAAGATCTGGGA (SEQ ID NO: 843)For screening deletion of sep1 ORF T1059_serendo_orf_probef2CTTCTGCAAGGAGGACGATT (SEQ ID NO: 844) T1060_serendo_orf_prober2GCGATGCCGTACGTGTAC (SEQ ID NO: 845)

Example 3 Generation of 11-Fold Protease Deletion Strain with slp8Deletion (M750)

The deletion plasmid pTTv330 for subtilisin-like protease slp8(tre58698) is described in Example 12 of WO2013/102674.

To generate a 11-fold protease deletion strain, removal of the pyr4marker was applied to the 10-fold deletion strain M633 essentially asdescribed above. Consecutive 5-FOA selection steps were carried out toensure that the clones selected were originating from single cells.

Final clones were verified by PCR using the primers listed in Table 3-1.Signal corresponding to successful removal of the blaster cassette wasobtained for majority of the clones. Removal of the blaster cassette wasfurther verified by plating the clones onto minimal medium plates withor without 5 mM uridine. and resulting strain used in generation of11-fold protease deletion strain was designated with strain number M637(clone 1-G).

To remove vector sequence, plasmid pTTv330 (Δslp8-pyr4-hph) was digestedwith MssI+SbfI and the correct fragment purified from an agarose gelusing a QIAquick Gel Extraction Kit (Qiagen). Approximately 5 μg of thedeletion cassette was used to transform 10-fold protease deletion strainM637 (Δpep1Δtsp1Δslp1Δgap1Δgap2Δpep4Δpep3Δpep5Δpep2Δsep1, pyr4−).Preparation of protoplasts and transformation were carried outessentially as described above.

Transformants were picked as first streaks. Growing streaks werescreened by PCR (using the primers listed in Table 3-1) for correctintegration and clones giving the expected signals were purified tosingle cell clones and rescreened by PCR using the primers listed inTable 3-1. Deletion of slp8 was verified by Southern analyses fromselected clones (FIG. 2 A, B, C) using methods described above. Clone54-131-2 was designated with strain number M750.

TABLE 3-1 Primers for screening removal of pyr4blaster cassette from 10-fold protease deletionstrain and for screening pTTv330/Δslp8-pyr4-hphintegration and strain purity. Primer SequenceFor screening removal of pyr4 blastercassette from M633 and strain purity T1173_sep1_5f_f2TAGGTCTCGGCTGACAAGG (SEQ ID NO: 846) T1174_sep1_3f_r2GCTTCTTCCACTGAATGCTC (SEQ ID NO: 847) T488_pyr4_5utr_revGGAGTTGCTTTAATGTCGGG (SEQ ID NO: 428) T1059_serendo_orf_probef2CTTCTGCAAGGAGGACGATT (SEQ ID NO: 844) T1060_serendo_orf_prober2GCGATGCCGTACGTGTAC (SEQ ID NO: 845) For screening integration ofpTTv330 (Δslp8-pyr4-hph) T1298_slp8_5int CATCACCAAGAAGGTCCCTC(SEQ ID NO: 848) T488_pyr4_5utr_rev GGAGTTGCTTTAATGTCGGG(SEQ ID NO: 428) T061_pyr4_orf_screen_2F TTAGGCGACCTCTTTTTCCA(SEQ ID NO: 382) T1299_slp8_3int ACTCAATGGAACCACATCGA (SEQ ID NO: 849)For screening deletion of slp8 ORF T1300_slp8_orf_forCTGTGGTTGAGTGCAGATG (SEQ ID NO: 850) T1301_slp8_orf_revTCCCACACATCAACACAAGT (SEQ ID NO: 851)

Example 4 Generation of 12-Fold Protease Deletion Strain with amp2Deletion (M893)

To generate a 12-fold protease deletion strain, removal of the pyr4-hphdouble marker was applied to the 11-fold deletion strain M750essentially as described above. Consecutive 5-FOA selection steps werecarried out to ensure that the clones selected were originating fromsingle cells.

Final clones were verified by PCR using the primers listed in Table 4-1.Signal corresponding to successful removal of the blaster cassette wasobtained for majority of the clones. Removal of the blaster cassette wasfurther verified by plating the clones onto minimal medium plates withor without 5 mM uridine. In addition, clones were tested also onhygromycin plates with or without 5 mM uridine. No growth was observedon the plates without uridine supplementation or if plates containedhygromycin. Resulting marker-free strain used in generation of 12-foldprotease deletion strain was designated with strain number M780 (clone1AH).

To remove vector sequence, plasmid pTTv327 (Δamp2-pyr4-hph) was digestedwith MssI+SbfI and the correct fragment purified from an agarose gelusing a QIAquick Gel Extraction Kit (Qiagen). More than 6 μg of thedeletion cassette was used to transform 11-fold protease deletion strainM780 (Δpep1Δtsp1Δslp1Δgap1Δgap2Δpep4Δpep3Δpep5Δpep2Δsep1Δslp8, pyr4−,hph−). Preparation of protoplasts and transformation were carried outessentially as above.

Transformants were picked as first streaks. Growing streaks werescreened by PCR (using the primers listed in Table 4-1) for correctintegration. Clones giving the expected signals were purified to singlecell clones and rescreened by PCR using the primers listed in Table 4-1.Deletion of amp2 was verified by Southern analyses from selected clones(FIG. 3 A, B, C). Clone 59-6A was designated with strain number M893.

TABLE 4-1 Primers for screening removal of pyr4-hphblaster cassette from 11-fold proteasedeletion strain and for screening pTTv327/Δamp2-pyr4-hph integration and strain purity. Primer SequenceFor screening removal of pyr4-hph blastercassette from M750 and strain purity T047_trpC_term_end_FCCTATGAGTCGTTTACCCAGA (SEQ ID NO: 426) T1299_slp8_3intACTCAATGGAACCACATCGA (SEQ ID NO: 849) T061_pyr4_orf_screen_2FTTAGGCGACCTCTTTTTCCA (SEQ ID NO: 382) T624_gpdA_seqR1CTCCATATTCTCCGATGATGC (SEQ ID NO: 852) T1420_slp8_5f_f2TGAATTTGTTGGATCCCTGC (SEQ ID NO: 853) T1421_slp8_3f_r2TACCCAGGTCAAAAGAGCAG (SEQ ID NO: 854) T1292_Hygrorf_ForGCCTGAACTCACCGCGACG (SEQ ID NO: 855) T1293_Hygrorf_RevCCTTTGCCCTCGGACGAGTG (SEQ ID NO: 856) For screening integration of pTTv327 (Δamp2-pyr4-hph) T1083_amp2_screen_5flk_fwd CCACTGAAGGGGAGTTTTCA(SEQ ID NO: 857) T488_pyr4_5utr_rev GGAGTTGCTTTAATGTCGGG(SEQ ID NO: 428) T047_trpC_term_end_F CCTATGAGTCGTTTACCCAGA(SEQ ID NO: 426) T1085_amp2_screen_3flk_rev TCGCGGTATCGTATGAGATG(SEQ ID NO: 858) For screening deletion of amp2 ORF T1086_amp2_orf_fwdGCCAGCTTCAACATCGACTT (SEQ ID NO: 859) T1087_amp2_orf_revCAGCACGAGCACGTTGTACT (SEQ ID NO: 860)

Example 5 The 13-Fold Protease Deletion Strain Having DeletionsΔpep1Δtsp1Δslp1Δgap1Δgap2Δpep4Δpep3Δpep5Δpep2Δsep1Δslp8Δamp2Δslp7

To generate a 13-fold protease deletion strain, removal of the pyr4-hphdouble marker was applied to the 12-fold deletion strain M893 (59-6A,pTTv327 in M780) as described above. Consecutive 5-FOA selection stepswere carried out to ensure that the clones selected were originatingfrom single cells.

Final clones were verified by PCR using the primers listed in Table 5-1.Signal corresponding to successful removal of the blaster cassette wasobtained for majority of the clones. Removal of the blaster cassette wasfurther verified by plating the clones onto minimal medium plates withor without 5 mM uridine. In addition, clones were tested also onhygromycin plates with or without 5 mM uridine. No growth was observedon the plates without uridine supplementation or if plates containedhygromycin. Resulting marker-free strain used in generation of 13-foldprotease deletion strain was designated with strain number M901 (clone1A2).

To remove vector sequence, plasmid pTTv329 (Δslp7-pyr4-hph) was digestedwith MssI+SbfI and the correct fragment purified from an agarose gel.Approximately 10 μg of the deletion cassette was used to transform12-fold protease deletion strain M901 (Δpep1Δtsp1Δslp1Δgap1Δgap2Δpep4Δpep3Δpep5Δpep2Δsep1 Δslp8Δamp2, pyr4−, hph−).Preparation of protoplasts was carried out as above. Transformation wascarried out using modified transformation method for protoplasts. Inthis method DNA was introduced to the protoplasts as described above butmedia and method for plating was changed. Transformation plates andfirst top agar were potato dextrose agar medium containing 1 M sorbitolfor osmotic stabilisation. Protoplasts were added to the first top agar,mixed, poured onto the PD agar plates and let to regenerate for a few(2-4) hours at room temperature. After the short regeneration, a secondtop agar containing 10 g/l sorbose, 10 g/l cellobiose, 10 g/l yeastextract, 1 M sorbitol, 5 g/l (NH4)2SO4 (+agar & salts) and 150 μg/mlHygromycin B was poured on top of the first top agar.

Transformants were picked as first streaks. Growing streaks werescreened by PCR (using the primers listed in Table 5-1) for correctintegration. Clones giving the expected signals were purified to singlecell clones and rescreened by PCR using the primers listed in Table 5-1.

TABLE 5-1 Primers for screening removal of pyr4-hphblaster cassette from 12-fold proteasedeletion strain and for screening pTTv329/Δslp7-pyr4-hph integration and strain purity. Primer SequenceFor screening removal of pyr4-hph blastercassette from M893 and strain purity T1556_amp2_5f_f2AAGTGTGCTGATGTGATGGA (SEQ ID NO: 861) T1557_amp2_3f_r2GCATGCGAAGTACCTTACGA (SEQ ID NO: 862) T061_pyr4_orf_screen_2FTTAGGCGACCTCTTTTTCCA (SEQ ID NO: 382) T624_gpdA_seqR1CTCCATATTCTCCGATGATGC (SEQ ID NO: 852) For screening integration ofpTTv329 (Δslp7-pyr4-hph) T1092_slp7_screen_5flk_fwd TTGGTTTGAACAGCTGCAAG(SEQ ID NO: 677) T488_pyr4_5utr_rev GGAGTTGCTTTAATGTCGGG(SEQ ID NO: 428) T047_trpC_term_end_F CCTATGAGTCGTTTACCCAGA(SEQ ID NO: 426) T1093_slp7_screen_3flk_rev ATGGTCAGCCAGAACCTGAC(SEQ ID NO: 680) For screening deletion of slp7 ORF T1094_slp7_orf_fwdTCTTGAGCCGTTTCTCGAAT (SEQ ID NO: 681) T1095_slp7_orf_revCCGCTCTTAGATCGATGGTC (SEQ ID NO: 682)

Example 6 Generation of 13-Fold Protease Deletion Strain with slp3

To generate a 13-fold protease deletion strain, removal of the pyr4-hphdouble marker was applied to the 12-fold deletion strain M893 asdescribed above. Consecutive 5-FOA selection steps were carried out toensure that the clones selected were originating from single cells.

Final clones were verified by PCR using the primers listed in Table 6-1.Signal corresponding to successful removal of the blaster cassette wasobtained for majority of the clones. Removal of the blaster cassette wasfurther verified by plating the clones onto minimal medium plates withor without 5 mM uridine. In addition, clones were tested also onhygromycin plates with or without 5 mM uridine. Resulting marker-freestrain used in generation of 13-fold protease deletion strain wasdesignated with strain number M901 (clone 1A2).

To remove vector sequence, plasmid pTTv425 (Δslp3-pyr4) was digestedwith MssI and the correct fragment purified from an agarose gel.Approximately 5 μg of the deletion cassette was used to transform12-fold protease deletion strain M901 (Δpep1Δtsp1Δslp1Δgap1Δgap2Δpep4Δpep3Δpep5Δpep2Δsep1Δslp8Δamp2, pyr4−, hph−).Preparation of protoplasts and transformation were carried out as above.Transformants were picked as first streaks. Growing streaks werescreened by PCR (using the primers listed in Table 6-1) for correctintegration and clones giving the expected signals were purified tosingle cell clones and rescreened by PCR using the primers listed inTable 6-1.

TABLE 6-1 Primers for screening removal of pyr4-hphblaster cassette from 12-fold protease deletion strain and for screeningpTTv425/Δslp3-pyr4 integration and strain purity. Primer SequenceFor screening removal of pyr4-hph blastercassette from M893 and strain purity T1556_amp2_5f_f2AAGTGTGCTGATGTGATGGA (SEQ ID NO: 861) T1557_amp2_3f_r2GCATGCGAAGTACCTTACGA (SEQ ID NO: 862) T061_pyr4_orf_screen_2FTTAGGCGACCTCTTTTTCCA (SEQ ID NO: 382) T624_gpdA_seqR1CTCCATATTCTCCGATGATGC (SEQ ID NO: 852) For screening integration ofpTTv425 (Δslp3-pyr4) T056_slp3_5screen_F GTGAATGGGTGGCAACATGA(SEQ ID NO: 333) T488_pyr4_5utr_rev GGAGTTGCTTTAATGTCGGG(SEQ ID NO: 428) T061_pyr4_orf_screen_2F TTAGGCGACCTCTTTTTCCA(SEQ ID NO: 382) T057_slp3_3screen_R CATCAAGTTGACCACCATTGT(SEQ ID NO: 336) For screening deletion of slp3 ORFT1581_slp3_ORF_probe2_f AGTTAATGATGCCCGTCTTG (SEQ ID NO: 663)T1582_slp3_ORF_probe2_r GAGCGTCTCCTGTTAGCTTG (SEQ ID NO: 664)

Example 7 Generation of 13-Fold Protease Deletion Strain with pep9

To generate a 13-fold protease deletion strain, removal of the pyr4-hphdouble marker was applied to the 12-fold deletion strain M893essentially as described above. Consecutive 5-FOA selection steps werecarried out to ensure that the clones selected were originating fromsingle cells.

Final clones were verified by PCR using the primers listed in Table 7-1.Signal corresponding to successful removal of the blaster cassette wasobtained for majority of the clones. Removal of the blaster cassette wasfurther verified by plating the clones onto minimal medium plates withor without 5 mM uridine. In addition, clones were tested also onhygromycin plates with or without 5 mM uridine. Resulting marker-freestrain used in generation of 13-fold protease deletion strain wasdesignated with strain number M901 (clone 1A2).

To remove vector sequence, plasmid pTTv426 was digested with MssI andthe correct fragment purified from an agarose gel. Approximately 5 μg ofthe deletion cassette was used to transform 12-fold protease deletionstrain M901 (Δpep1Δtsp1Δslp1Δgap1 Δgap2Δpep4Δpep3Δpep5Δpep2Δsep1Δslp8Δamp2, pyr4−, hph−). Preparation of protoplasts and transformationwere carried out as above. Transformants were picked as first streaks.Growing streaks were screened by PCR (using the primers listed in Table7-1) for correct integration and clones giving the expected signals werepurified to single cell clones and rescreened by PCR using the primerslisted in Table 7-1.

TABLE 7-1 Primers for screening removal of pyr4-hphblaster cassette from 12-fold proteasedeletion strain and for screening pTTv426/Δpep9-pyr4 integration and strain purity. Primer SequenceFor screening removal of pyr4-hph blastercassette from M893 and strain purity T1556_amp2_5f_f2AAGTGTGCTGATGTGATGGA (SEQ ID NO: 861) T1557_amp2_3f_r2GCATGCGAAGTACCTTACGA (SEQ ID NO: 862) T061_pyr4_orf_screen_2FTTAGGCGACCTCTTTTTCCA (SEQ ID NO: 382) T624_gpdA_seqR1CTCCATATTCTCCGATGATGC (SEQ ID NO: 852) For screening integration ofpTTv426 (Δpep9-pyr4) T1031_pep9_screen_5flk_fwd GGGTTGGAGATGTTGGAAGA(SEQ ID NO: 865) T488_pyr4_5utr_rev GGAGTTGCTTTAATGTCGGG(SEQ ID NO: 428) T061_pyr4_orf_screen_2F TTAGGCGACCTCTTTTTCCA(SEQ ID NO: 382) T1032_pep9_screen_3flk_rev TTGACGAGACGGGGAGTTAC(SEQ ID NO: 866) For screening deletion of pep9 ORF T1033_pep9_orf_fwdCAGCCCTGACACCACTCTCT (SEQ ID NO: 867) T1034_pep9_orf_revTCCAGTCCTTGGGAGAAATG (SEQ ID NO: 868)

Example 8 Generation of 9-Fold Protease Deletion Strains from theInterferon Producing Strain M577

Generation of 9-Fold Deletion Strain with amp1 Deletion

To remove the deletion cassette, plasmid pTTv240 (Δamp1-pyr4-hph) wasdigested with PmeI and the correct fragment was purified using aQIAquick Gel Extraction Kit (Qiagen). Approximately 5 μg of the deletioncassette was used to transform the 8-fold protease deletion strain M577(Δpep1Δtsp1 Δslp1Δgap1Δgap2Δpep4Δpep3Δpep5; see Example 18 ofWO2013/102674), which produces interferon alpha 2b. Preparation ofprotoplasts and transformation were carried out essentially as describedabove using hygromycin selection. Transformants were picked and streakedon selection plates. Growing streaks were screened by PCR (using theprimers listed in Table 8-1) for correct integration. Transformantsgiving the expected signals were purified to single cell clones andrescreened by PCR using the primers listed in Table 8-1. The clone 108Awas designated with strain number M669.

TABLE 8-1 Primers for screening pTTv240/Δamp1-pyr4-hghintegration and strain purity. Primer SequenceFor screening integration of pTTv240 (Δamp1-pyr4-hph)T840_amp1_screen_5 flk fwd TGGCATTGATCTAGAACCTCCT (SEQ ID NO: 869)T1084_screen_5flk_pyr_rev TCTTGAGCACGACAATCGAC (SEQ ID NO: 662)T1015_screen_3flk_hygro_fwd GCATGGTTGCCTAGTGAATG (SEQ ID NO: 663)T843_amp1_scrn_rev 3 flk GACGACTTGGTGGAGCTCAT (SEQ ID NO: 870)For screening deletion of amp1 ORF T940_amp1_orf2_fwGACTACCCCCAGAACGTCAA (SEQ ID NO: 871) T941_amp1_orf2_revAAGAGGCGGATCTTTTGGTT (SEQ ID NO: 872)Generation of 9-Fold Deletion Strain with slp7 Deletion

The deletion plasmid pTTv269 for the serine protease slp7 (tre123865) isdescribed in Example 11 of WO2013/102674.

To remove the deletion cassette, plasmid pTTv269 (Δslp7-pyr4-hph) wasdigested with PmeI and the correct fragment was purified using aQIAquick Gel Extraction Kit (Qiagen). Approximately 5 μg of the deletioncassette was used to transform the 8-fold protease deletion strain M577(Δpep1Δtsp1 Δslp1Δgap1Δgap2Δpep4Δpep3Δpep5 see above). Preparation ofprotoplasts and transformation were carried out essentially as describedabove using hygromycin selection.

Transformants were picked and streaked on selection plates. Growingstreaks were screened by PCR (using the primers listed in Table 8-2) forcorrect integration. Transformants giving the expected signals werepurified to single cell clones and rescreened by PCR using the primerslisted in Table 8-2. The clone 5.64 was designated with strain numberM673.

TABLE 8-2 Primers for screening pTTv269/Δslp7-pyr4-hghintegration and strain purity. Primer SequenceFor screening integration of pTTv269 (Δslp7-pyr4-hgh)T1092_slp7_screen_5flk_fwd TTGGTTTGAACAGCTGCAAG (SEQ ID NO: 677)T1084_screen_5flk_pyr_rev TCTTGAGCACGACAATCGAC (SEQ ID NO: 662)T1015_screen_3flk_hygro_fwd GCATGGTTGCCTAGTGAATG (SEQ ID NO: 663)T1093_slp7_screen_3flk_rev ATGGTCAGCCAGAACCTGAC (SEQ ID NO: 680)For screening deletion of slp7 ORF T1094_slp7_orf_fwdTCTTGAGCCGTTTCTCGAAT (SEQ ID NO: 681) T1095_slp7_orf_revCCGCTCTTAGATCGATGGTC (SEQ ID NO: 682)Generation of 9-Fold Deletion Strain with amp2 Deletion

To remove the deletion cassette, plasmid pTTv271 (Δamp2-pyr4-hgh) wasdigested with PmeI and the correct fragment was purified using aQIAquick Gel Extraction Kit (Qiagen). Approximately 5 μg of the deletioncassette was used to transform the 8-fold protease deletion strain M577.

Transformants were picked and streaked on selection plates. Growingstreaks were screened by PCR (using the primers listed in Table 8-3) forcorrect integration. Transformants giving the expected signals werepurified to single cell clones and rescreened by PCR using the primerslisted in Table 8-3. Clone 24A was designated with strain number M674.

TABLE 8-3 Primers for screening pTTv271/Δamp2-pyr4-hghintegration and strain purity. Primer SequenceFor screening integration of pTTv271 (Δamp2-pyr4-hph)T1083_amp2_screen_5flk_fwd CCACTGAAGGGGAGTTTTCA (SEQ ID NO: 857)T1084_screen_5flk_pyr_rev TCTTGAGCACGACAATCGAC (SEQ ID NO: 662)T1015_screen_3flk_hygro_fwd GCATGGTTGCCTAGTGAATG (SEQ ID NO: 663)T1085_amp2_screen_3flk_rev TCGCGGTATCGTATGAGATG (SEQ ID NO: 858)For screening deletion of amp2 ORF T1086_amp2_orf_fwdGCCAGCTTCAACATCGACTT (SEQ ID NO: 859) T1087_amp2_orf_revCAGCACGAGCACGTTGTACT (SEQ ID NO: 860)Generation of 9-Fold Deletion Strain with sep1 Deletion

To remove the deletion cassette, plasmid pTTv247 (Δsep1-pyr4-hgh) wasdigested with PmeI and the correct fragment was purified using aQIAquick Gel Extraction Kit (Qiagen). Approximately 5 μg of the deletioncassette was used to transform the 8-fold protease deletion strain M577.

Transformants were picked and streaked on selection plates. Growingstreaks were screened by PCR (using the primers listed in Table 8-4) forcorrect integration. Clones giving the expected signals were purified tosingle cell clones and rescreened by PCR using the primers listed inTable 8-4. Clone 47.1 was designated with strain number M668.

TABLE 8-4 Primers for screening pTTv247/Δsep1-pyr4-hgh integration and strain purity. Primer SequenceFor screening integration of pTTv247 (Δsep1-pyr4-hgh) T519_serendo_5intAACCACCTTGTTCTGTCCGT (SEQ ID NO: 842) T488_pyr4_5utr_revGGAGTTGCTTTAATGTCGGG (SEQ ID NO: 428) T521_serendo_3intGGAACTGTCAAGATCTGGGA (SEQ ID NO: 843) T1015_screen_3flk_hygro_fwdGCATGGTTGCCTAGTGAATG (SEQ ID NO: 663) For screening deletion of sep1 ORFT504_serendo_orf_probef GCCTCCGCCCTCCTCTTCCA (SEQ ID NO: 873)T505_serendo_orf_prober GCTTTGTCGAGCGGAGCGGT (SEQ ID NO: 874)Generation of 9-Fold Deletion Strain with pep9 Deletion

To remove the deletion cassette, plasmid pTTv267 (Δpep9-pyr4-hgh) wasdigested with PmeI and the correct fragment was purified using aQIAquick Gel Extraction Kit (Qiagen). Approximately 5 μg of the deletioncassette was used to transform the 8-fold protease deletion strain M577.

Transformants were picked and streaked on selection plates. Growingstreaks were screened by PCR (using the primers listed in Table 8-5) forcorrect integration. Clones giving the expected signals were purified tosingle cell clones and rescreened by PCR using the primers listed inTable 8-5. Transformant 77 was designated with strain number M671.

TABLE 8-5 Primers for screening pTTv267/Δpep9-pyr4-hgh integration andstrain purity. For screening integration of pTTv267 (Δpep9-pyr4-hgh)Primer Sequence T1031_pep9_screen_5flk_fwdGGGTTGGAGATGTTGGAAGA (SEQ ID NO: 865) T1084_screen_5flk_pyr_revTCTT GAGCACGACAATCGAC (SEQ ID NO: 662) T1015_screen_3flk_hygro_fwdGCATGGTTGCCTAGTGAATG (SEQ ID NO: 663) T1032_pep9_screen_3flk_revTTGACGAGACGGGGAGTTAC (SEQ ID NO: 866) For screening deletion of pep9 ORFT1033_pep9_orf_fwd CAGCCCTGACACCACTCTCT (SEQ ID NO: 867)T1034_pep9_orf_rev TCCAGTCCTTGGGAGAAATG (SEQ ID NO: 868)

Example 9 9-Fold Protease Deletion Strains Producing Interferon in 24Well Culture

The 9-fold protease deletion strains were grown in 24 well culture inTrMM with diammonium citrate without ammonium sulfate, 100 mM PIPPS, 20g/L spent grain extract, 40 g/L lactose at pH 4.5, shaking at 28° C.Immunoblotting was done to detect interferon alpha 2b. The supernatantwas diluted with water, so that 0.5 μl of each supernatant could beloaded into the 4-20% Criterion gel. Mixed with LSB+BME and heated at95° C. for 5 minutes. The proteins were transferred to nitrocellulosewith the Turbo semi-dry blotter for 7 minutes. The nitrocellulosemembrane was blocked with 5% milk in TBST for 1 hour. The interferonprotein was detected with a mouse anti-interferon alpha 2b antibody(Abcam #ab9386) diluted 0.5 μg/ml in TBST. The primary antibody wasincubated with the membrane for 1 hour shaking at room temperature. Theprimary antibody was removed and the membrane washed with TBST. Thesecondary antibody was goat anti-mouse AP conjugate (BioRadcat#170-6520) diluted 1:10,000 in TBST. The secondary antibody wasincubated for 1 hour shaking at room temperature, the antibody wasremoved, and membrane washed for 1 hour shaking at room temperature. Theblot was developed using AP substrate (Promega).

Observing the day 7 culture samples there was a dramatic effect on thestability of interferon. The slp7 and amp2 deletion strains continued toproduce interferon, while interferon was not stable in the M577 controland the amp1 and sep1 deletion strains. Results are shown in FIG. 4.

Example 10 9-Fold Protease Deletion Strains Producing Interferon inFermentor Culture

The 9-fold protease deletion strains expressing interferon werecultivated in fermentors as batch cultivations. They were grown in TrMMplus in 20 g/L yeast extract, 40 g/L cellulose, 80 g/L cellobiose, and40 g/L sorbose at pH 4.5 with the temperature shifting from 28° to 22°at 48h. These cultivations were done and assigned the culture codesFTR108_R1 (M668), FTR108_R2 (M669), FTR108_R6 (M673), and FTR108_R7(M674).

The supernatant was diluted in water and sample buffer so that 0.1 μlcould be loaded per well. The immunoblotting procedure to detectinterferon alpha 2b was carried out as described above with the 24 wellcultures.

The results of the fermentor cultivations can be seen in FIG. 5.Standard amounts representing 50, 100, and 200 ng of interferon wereused to generate a standard curve. The parental M577 control cultivationproduced 0.84 g/L of interferon on day 3. The M674 strain with the amp2protease deletion was the most improved strain overall providing 2.4 g/Lon day 3. Deletion of amp2 protease provided a 2.9 fold improvement ininterferon production. The slp7 protease deletion strain M673 achieved2.1 g/L on day 4. This was a 2.5 fold improvement over the parent strainM577. The sep1 protease deletion in M668 secreted 1.38 g/L ofinterferon. The pep9 deletion in strain M671 also improved theinterferon expression level to 1.03 g/L. The M669 strain with amp1deletion produced 0.36 g/L under these culture conditions (data notshown).

The strain M674 (Δamp2) was also cultivated with and without SBTIinhibitor addition. The medium was TrMM plus 20 g/L yeast extract, 40g/L cellulose, 80 g/L cellobiose, and 40 g/L sorbose at pH 4.5 with thetemperature shifting from 28° to 22° at 48h. The Triab 125 cultivationwas done without SBTI inhibitor and Triab 126 was done with SBTIinhibitor feeding (0.4 mg/ml target concentration).

The SBTI inhibitor improved the interferon expression level, see FIG. 6.The base level was 1.4 g/L on day 4, but with inhibitor treatment theinterferon expression could be increased to 4.5 g/L on day 5. Theaddition of inhibitor shifted the peak expression day until day 5, whichindicated higher stability of the interferon in the supernatant.

The supernatant was diluted in water so that 0.025 μl could be loaded in10 μl volume into a 4-20% SDS-PAGE gel Immunodetection done with Abcam(#ab9386) anti-IFN-α 2b antibody diluted to 1 μg/ml in TBST. Thesecondary antibody from Bio-rad (#170-6520) goat anti-mouse IgG APconjugated secondary antibody diluted 1:5000 in TBST. The proteinstandards were loaded in the gel corresponding 200 ng, 100 ng, and 50 ngof full length IFN-α 2b.

Example 11 Generation of 9-Fold Protease Deletion Strain M960 with pep2Deletion and slp2 RNAi from the Interferon Producing Strain M788 (pyr4−of M577)

The interferon production strain M577 had the pyr4 marker in the pep5locus, where the last protease deletion was made. To remove the pyr4loopout marker, the M577 strain was plated on 5FOA plates. The survivingcolonies were screened by PCR to check the presence and integration ofthe pyr4 marker. The clones that did not have the double marker by PCRwere tested on minimum medium agar plates with and without uridine.Those clones that could not grow on plates without uridine were selectedas marker loopout clones. One pyr4− negative clone was selected tobecome strain M788. Primers used in the screening are shown below intable 11-1.

TABLE 11-1 For screening integration of pyr4-hgh marker Primer SequenceT627_pep5_5int_new GTCGAAGATGTCCTCGAGAT (SEQ ID NO: 432)T488_pyr4_5utr_rev GGAGTTGCTTTAATGTCGGG (SEQ ID NO: 428)T061_pyr4_orf_screen_2F TTAGGCGACCTCTTTTTCCA (SEQ ID NO: 382)T628_pep5_3int_new TAGTCCATGCCGAACTGC (SEQ ID NO: 435)For screening deletion of marker T858_pep5_5f_f3GGAATCGTCACCAAGGAG (SEQ ID NO: 612) T755_pep5_3f_rev3CTTCTGGTGACATTCCGAC (SEQ ID NO: 613)

To remove the deletion cassette, the plasmids were digested with PmeIand the correct fragment was purified using a QIAquick Gel ExtractionKit (Qiagen). Approximately 5 μg of the deletion cassette was used totransform the 8-fold protease deletion strain M788 (Δpep1Δtsp1Δslp1Δgap1Δgap2Δpep4Δpep3Δpep5, pyr4−), which produces interferon alpha 2b.Preparation of protoplasts and transformation were carried outessentially as described above. The silencing cassettes were designed tointegrate into the pep2 locus (tre53961).

Transformants were picked and streaked on selection plates. Growingstreaks were screened by PCR (using the primers listed in Table 11-2)for correct integration. Clones giving the expected signals werepurified to single cell clones and rescreened by PCR using the primerslisted in Table 11-2. The transformation of pTTv376 produced a straindesignated as M960 and pTTv405 produced the M961 strain.

TABLE 11-2 Primers for screening pTTv376 and pTTv405/Δpep2-pyr4-hghintegration and strain purity.For screening integration of pTTv376 and pTTv405 (Δpep2-pyr4-hgh) PrimerSequence T596_pep2 fwd 5′flank screenCCTCTGCGTTGAGCAACATA (SEQ ID NO: 614) T624_gpdA_seqR1CTCCATATTCTCCGATGATGC (SEQ ID NO: 852) T600_pep2 rev 3′flank screenCGAAAGCGTGGAGTCTTCTC (SEQ ID NO: 615) T047_trpC_term_end_FCCTATGAGTCGTTTACCCAGA (SEQ ID NO: 426)For screening deletion of pep2 ORF T601_pep2 fwdGACGTGGTACGACAACATCG (SEQ ID NO: 269) T623_pep2 revTATCAAGGTACCGGGGACAG (SEQ ID NO: 270) Culturing in 24 well plates

Two transformants from the pTTv376 and pTTv405 transformations weregrown in 24 well cultures to compare their interferon production againstthe control strain M577. The strains were grown in TrMM with diammoniumcitrate without ammonium sulfate, 100 mM PIPPS, 20 g/L spent grainextract, 40 g/L lactose at pH 4.5, shaking at 28° C. Duplicate wellswere used for each transformant. Samples from the 24 well cultures takenon day 5 were used for immunoblotting. The supernatant was diluted withwater, so that 0.2 μl of each supernatant could be loaded into the 4-20%Criterion gel. Mixed with LSB+BME and heated at 95° C. for 5 minutes.The proteins were transferred to nitrocellulose with the BioRad Turbosemi-dry blotter for 7 minutes. The nitrocellulose membrane was blockedwith 5% milk in TBST for 1 hour. The interferon protein was detectedwith a mouse anti-interferon alpha 2b antibody (Abcam #ab9386) diluted0.5 μg/ml in TBST. The primary antibody was incubated with the membranefor 1 hour shaking at room temperature. The primary antibody was removedand the membrane washed with TBST. The secondary antibody goatanti-mouse IRDye 680RD conjugate (Li-cor #926-68070) diluted 1:30,000 inTBST. The secondary antibody was incubated for 1 hour shaking at roomtemperature. The secondary antibody was removed and membrane washed for1 hour in TBST before scanning the membrane. The membrane was scanned at700 nm using the Odyssey CLx near infrared imaging system (Li-cor,Inc.).

The 24 well cultivation results can be seen in FIG. 7. Both strains withslp2 silencing produced high amounts of interferon. The best pTTv405transformant 405b produced 427 mg/L of interferon which was assignedstrain number M960. The best pTTv376 transformant 376a produced up to534 mg/L of interferon and was called strain M961. The control strain(M577) reached up to 200 mg/L. The M961 strain produced 2.7 times moreinterferon in this cultivation and 2.1 times more with M960 compared tocontrol strain, respectively.

Fermentation

The two strains were cultivated in 1 L fermentors as batch cultures inTrMM in 20 g/L yeast extract, 40 g/L cellulose, 80 g/L cellobiose, and40 g/L sorbose at pH 4.5 with the temperature shifting from 28° to 22°at 48h. Triab152 and Triab153 cultivations were done with strains M960and M961, respectively. The supernatant samples were diluted in waterand loading dye so that 0.05 μl of supernatant was loaded per wellImmunoblotting for detection was done as described above for interferon.Standard amounts of interferon representing 400, 200. 100, 50 and 25 ngwere used to construct a standard curve to determine expressedconcentration.

In cultivation Triab152 the M960 strain produced 1.4 g/L on day 3 (FIG.8) and the M577 strain produced 1.2 g/L on day 3 (data not shown). Thestrain M961 achieved 3.2 g/L on day 4 which was 2.7 times moreinterferon than the parental strain M577.

Example 12 Introduction of Heterologous Proteins into the ProteaseDeletion Strains

To generate the MAB01 antibody producing T. reesei strain in any of theprotease deleted strain(s) of the invention a strain is transformed withMAB01 light and heavy chain constructs (pTTv98, pTTv99, pTTv67, pTTv101,pTTv102, and/or pTTv223, see also Examples 1-3 of WO2013/102674) usinghygromycin and acetamide in selection as described above. To producerituximab antibody, constructs having harmonised heavy chain fused withCBHI carrier and light chain fused with CBHI carrier are transformed toprotease deletion strains of T. reesei as described above.

To express various antibody fragments (Fabs, multimeric single domainantibodies (sd-Ab's) and scFVs) in different protease deletionbackgrounds of the invention methods of Example 23 are applicable. Thearchitecture of the genetic expression cassettes applied for thispurpose is usually based on the regulatory elements (promoter andterminator) of the cellobiohydrolase I (cbh1) gene. The catalytic domainof the CBHI protein is modified to remove intron sequences and used asfusion partner to enhance antibody fragment expression and secretion. Arecognition motif for the Kex2 protease is inserted in between thefusion partners to promoter co-secretory release of the antibodyfragments from the CBHI carrier protein and the expression cassettes areflanked by homologous regions to allow targeted integration to the T.reesei cbh1 locus.

T. reesei protease deletion strains are transformed with the purifiedexpression cassettes as described and selected for appropriate selectionmarker. Transformants are screened by PCR for homologous integration ofthe expression cassette to the cbh1 locus using a forward primer outsidethe 5′ flanking region fragment of the construct and the reverse primerinside the modified CBHI catalytic domain (5′ integration) as well as aforward primer inside the selection marker gene(s), respectively, and areverse primer outside the 3′ flanking region fragment (3′ integration).Proper integration of the disruption cassette is reconfirmed by PCRusing the same primer combinations as described above and the absence ofthe parental CBHI locus is verified by using a primer combinationtargeted to the cbh1 open reading frame. Correct integration of thedisruption cassette is additionally verified for all clones applyingSouthern hybridization.

Transformed T. reesei strains are cultivated in fermentation and shakeflask conditions as described earlier. Samples are collected in thecourse of the cultivations, and production levels are analysed, forexample, by affinity liquid chromatography as described above. Thequality of the purified samples is checked by SDS-PAGE.

Example 13 Glycoengineering of the Protease Deletion Strains

Generation of G0 Producing Strains

The generation of alg3 deletion plasmids pTTg156 (human full length GnT1and human full length GnT2) and pTTg173 (Kre2 N-terminal fusion withcatalytic domain of human GnT1 and full length human GnT2) are describedin Example 19 of WO2013/102674.

A T. reesei MAB01 expression strain is transformed with the PmeIfragments of pTTg156 and pTTg173. Variable amount of transformants(100-170 depending on the construct) are picked onto selective platesand on the basis of PCR screening clones with positive resultsconcerning 5′- and 3′-integration are selected for single spore platingsand re-screening for integration and alg3 deletion using primers asdescribed in Table 19.3 of Example 19 of WO2013/102674. PCR-screenedstrains are subjected to shake flask and fermentation cultivations,samples are obtained in appropriate days and the samples are subjectedto antibody concentration and glycan analyses.

Generation of GlcNacMan5 Producing Strains

The generation of plasmids for fusion proteins of targeting peptide andcatalytic domain of human GnT1 plasmids pTTv274 (N-terminal portion ofhuman GnT2), pTTv275 (N-terminal portion of T. reesei Kre2), and pTTv278(N-terminal portion of T. reesei Och1) are described in Example 21 ofWO2013/102674.

Fragments for transformations are released from the above plasmids withPmeI. All fragments are transformed individually to a MAB01 (or anantibody such as rituximab) expressing strain and protoplasttransformations are carried out essentially as described above.

Well growing clones on selective streaks are screened for the 5′ and 3′integration into the egl2 locus. Double integration-positive clones areadditionally screened for the loss of the egl2 ORF. The clones givingthe desired results are purified through single spore platings, and thesingle spore-derived clones are verified by PCR to be pure integrationstrains. Selected strains are subjected to shake flask and fermentationcultivations, samples are obtained in appropriate days and the samplesare subjected to antibody concentration and glycan analyses.

For N-glycan analysis MAB01 is purified from culture supernatants usingProtein G HP MultiTrap 96-well filter plate (GE Healthcare) according tomanufacturer's instructions and the antibody concentrations aredetermined via UV absorbance against MAB01 standard curve. N-glycans arereleased from EtOH precipitated and SDS denatured antibody using PNGaseF (ProZyme Inc.) in 20 mM sodium phosphate buffer, pH 7.3, in overnightreaction at +37° C. The released N-glycans are purified with HypersepC-18 and Hypersep Hypercarb (Thermo Scientific) and analysed withMALDI-TOF MS.

Example 14 Protease Homologs

T. reesei sep1, amp1, amp2, and pep9 homologs were identified from otherorganisms.

BLAST searches were conducted using the National Center forBiotechnology Information (NCBI) non-redundant amino acid database usingthe Trichoderma reesei protease amino acid sequences as queries.Trichoderma virens and Trichoderma atroviride BLAST searches wereconducted using the DOE Joint Genome Institute's web site (Trichodermavirens Gv29-8 v2.0 and Trichoderma atroviride v2.0, respectively).Sequence hits from the BLAST searches were aligned using the ClustalW2or Clustal Omega alignment tool provided by EBI. Phylogenetic trees werealso generated using the sequence alignments.

FIG. 9 depicts a phylogenetic tree of amp1 and amp2 of selectedfilamentous fungi.

FIG. 10 depicts a phylogenetic tree of sep1 of selected filamentousfungi.

FIG. 11 depicts a phylogenetic tree of pep9 of selected filamentousfungi.

Example 15 Protease Activity Measurement of Protease Deficient T. reeseiStrains

The protein concentrations were determined from supernatant samples fromday 2-7 of 1×-7× protease deficient strains according to EnzChekprotease assay kit (Molecular probes #E6638, green fluorescent caseinsubstrate). Briefly, the supernatants were diluted in sodium citratebuffer to equal total protein concentration and equal amounts of thediluted supernatants were added into a black 96 well plate, using 3replicate wells per sample. Casein FL diluted stock made in sodiumcitrate buffer was added to each supernatant containing well and theplates were incubated covered in plastic bag at 37° C. The fluorescencefrom the wells was measured after 2, 3, and 4 hours. The readings weredone on the Varioskan fluorescent plate reader using 485 nm excitationand 530 nm emission. Some protease activity measurements were performedusing succinylated casein (QuantiCleave protease assay kit, Pierce#23263) according to the manufacturer's protocol.

The pep1 single deletion reduced the protease activity by 1.7-fold, thepep1/tsp1 double deletion reduced the protease activity by 2-fold, thepep1/tsp1/slp1 triple deletion reduced the protease activity by3.2-fold, the pep1/tsp1/slp1/gap1 quadruple deletion reduced theprotease activity by 7.8-fold compared to the wild type M124 strain, thepep1/tsp1/slp1/gap1/gap2 5-fold deletion reduced the protease activityby 10-fold, the pep1/tsp1/slp1/gap1/gap2/pep4 6-fold deletion reducedthe protease activity by 15.9-fold, and thepep1/tsp1/slp1/gap1/gap2/pep4/pep3 7-fold deletion reduced the proteaseactivity by 18.2-fold.

The FIG. 20 graphically depicts normalized protease activity data fromculture supernatants from each of the protease deletion supernatants(from 1-fold to 7-fold deletion mutant) and the parent strain M124.Protease activity was measured at pH 5.5 in first 5 strains and at pH4.5 in the last three deletion strains. Protease activity is againstgreen fluorescent casein. The six-fold protease deletion strain has only6% of the wild type parent strain and the 7-fold protease deletionstrain protease activity was about 40% less than the 6-fold proteasedeletion strain activity.

Example 16 IGF1 Production in the 13-Fold Protease Deletion Strain

An IGF1 expression cassette was created from a plasmid, which containedcbh1 promoter, terminator and 3′flank and amdS marker, to express IGF1as a fusion protein with CBHI carrier and without any carrier cleavagesites or purification tags. The construct was designed to be integratedinto the cbh1 locus under control of the native cbh1 promoter andterminator. The expression construct was cut out of the vector with PmeIand purified via standard methods. The Trichoderma transformation wasmade into the 13-fold protease deletion strain M1076 using AmdSselection marker (M1076 was generated from M901 by deleting pep9(tre79807)). Transformants were screened by PCR for loss of cbh1 orf andproper integration of the expression cassette into the locus.

The CBHI carrier sequence: SEQ ID NO. 924.

The IGF1 protein sequence expressed: SEQ ID NO. 925.

The M1140 was grown in the fermentator cultivation T189 without the useof protease inhibitors in TrMM with 20 g/L yeast extract, 40 g/Lcellulose, 100 g/L cellobiose, 40 g/L sorbose at pH 4.5 in a 1 literfermentor. The temperature was shifted from 28° C. to 22° C. after 48hours.

The expression level was checked via immunoblotting. The samples werediluted in orange LSB so that 0.0125 μl of supernatant could be loadedper well in a 4-20% Criterion gel. Immunoblotting was done to dualdetect the IGF1 and CBHI expression. Detection was done with rabbitanti-IGF1 diluted to 0.25 μg/ml in TBST (Abcam rabbit polyclonalantibody, ab9572) and anti-CBHI mab261 diluted 1:10,000 in TBST. Thesecondary antibody was goat anti-rabbit IgG 1:30,000 dilution in TBST(Li-Cor #926-68071, IRDye 680RD goat anti-rabbit IgG). The anti-mouseCBHI antibody was detected with Li-Cor #926-32210 IRDye 800CW goatanti-mouse IgG diluted 1:30,000 in TBST. Washed for 1 hour in TBST andrinsed with TBS. The filters were scanned with LI-COR Odyssey CLxInfrared Imaging System at 700 nm and 800 nm The expression of IGF1 wasmeasured from 44 hours to 162 hours compared to an IGF1 standard curve.The expressed fusion protein was around 70 kD. At 71 hours theexpression level was 3.5 g/L. The highest expression level measured was7.9 g/L at 92 hours. At 116 hours the expression level reduced to 6.6g/L. The IGF1 expression reduced significantly after 116 hours in thisbatch cultivation. The CO₂ peak was around 92 hours, which correlatedwell with the expression peak. After 116 hours the IGF1 protein wasdegraded off the carrier and only CBHI protein could be seen in thesamples. CBHI expression could be observed from 71 hours until 162hours. No protease inhibitors were used in this cultivation. The 13protease deletions improved the stability of the IGF1. Proteaseinhibitors may be needed to reach higher production levels in strainshaving less protease deletions. With M1140 it would be possible toachieve higher production levels if more cellulose was used in thecultivation.

The M231 production strain was made to produce a CBHI-TEV site-IGF1fusion protein in a strain with no protease deletions, M124. The TEVprotease cleavage site was included in between the CBHI carrier andIGF1. IGF1 was very stable and remained predominately in the fusion formafter secretion into the supernatant. The expression construct containedthe CBHI carrier-TEV cleavage site-IGF1 protein sequence and wasdesigned to be integrated into the CBHI locus under control of thenative promoter and terminator. After transformation transformants werescreened by PCR for loss of cbh1 orf and proper integration of theexpression cassette into the cbh1 locus. The final transformant wasnamed M231.

CBHI carrier sequence used to create M231 was the same as for IGF1 aboveexcept after C-terminal amino acids “ . . . TTTGSS” of the CBH1 aminoacids “PGP” were included before TEV cleavage site (ENLYFQ).

A second strain that produced an IGF1 variant called BVS857 was made ina similar way as M231 but the expression construct included a strepIItag and spacer before the TEV cleavage site. The expression cassette wasintegrated into the cbh1 locus under control of the native cbh1 promoterand terminator. The vector was digested with PmeI, the expressioncassette was gel purified, and was transformed into the M194 strain andselected for using AmdS selection. Transformants were screened by PCRfor loss of cbh1 orf and proper integration of the expression cassetteinto the cbh1 locus. The resulting strain M236 strain produced aCBHI-streptII tag-3×(GGGS)-TEV site-BVS857 fusion protein. The strepIItag was added to allow for purification, which could be convenientlyremoved afterward with TEV protease treatment.

The IGF1 strain M231 has no protease deletions, while the BVS857 strainM236 has the pep1 and tsp1 protease deletions. Both these strains wereexpressed as stable CBHI-IGF1 fusions into the supernatant. The M231 andM236 were cultivated in 1 L fermentors in the presence of chymostatin(20 μM) and pepstatin (10 μM) inhibitors. Both strains were cultivatedin TrMM with 20 g/L yeast extract, 40 g/L cellulose, 100 g/L cellobiose,40 g/L sorbose at pH 4.5 with chymostatin and pepstatin inhibitors addedon day 3, 4, and 5. The temperature was shifted from 28° C. to 22° C.after 48 hours.

The production levels were assessed by immunoblotting. The M231 culturesupernatant was diluted with orange LSB so that 0.025 μl could be loadedinto a 10 μl volume into a 4-20% criterion gel with IGF1 standards. TheM236 culture supernatant was diluted so that 0.05 μl of supernatant wasloaded to each well along with BVS857 standards onto a 4-20% criteriongel. Both blots were detected with rabbit anti-IGF1 diluted to 0.25μg/ml in TBST (Abcam rabbit polyclonal antibody, ab9572) and anti-CBHImab261 diluted 1:10,000 in TBST. The secondary antibody was goatanti-rabbit IgG 1:30000 dilution in TBST (Li-Cor #926-68071, IRDye 680RDgoat anti-rabbit IgG). The anti-mouse CBHI antibody was detected withLi-Cor #926-32210 IRDye 800CW goat anti-mouse IgG diluted 1:30,000 inTBST. Washed for 1 hour in TBST and rinsed with TBS. The filters werescanned with Li-Cor Odyssey CLx Infrared Imaging System at 700 nm and800 nm

The strain M231 demonstrated that up to 19 g/L of IGF1 could be producedas a fusion to CBHI carrier at 118 hours. The fusion protein ran around75 kD on the immunoblot. The CBHI-IGF1 fusion protein expression levelwas 2 g/L at 72 hours, 10 g/L at 95 hours, 16 g/L at 101 hours, and 19g/L at 118 hours. The CBHI expression level accumulated throughout thebatch cultivation from 49 hours to 118 hours.

With strain M236 up to 7 g/L of the BVS857 variant could be produced asa CBHI carrier fusion at 101 hours. The fusion protein ran around 75 kDon the immunoblot. The CBHI-BVS857 fusion protein expression level was2.7 g/L at 72 hours, 6.2 g/L at 95 hours, 7.0 g/L at 101 hours, 5.1 g/Lat 118 hours, and 2.5 g/L at 140 hours. As a fusion with CBHI carrierboth these IGF1 proteins are stably expressed into the supernatant.

The M236 IGF1-BVS857 productions strain was further cultivated infermentors with and without chymostatin and pepstatin inhibitors to seethe effect of proteases. This strain was cultivated previously withinhibitors and using 40 g/L cellulose. The M236 strain was cultivated inTrMM with 20 g/L yeast extract, 80 g/L cellulose, 100 g/L cellobiose, 40g/L sorbose at pH 4.5 with and without chymostatin and pepstatininhibitors added on day 3, 4, and 5. The temperature was shifted from28° C. to 22° C. after 48 hours.

No CBHI-BVS857 fusion protein or free protein could be detected in thesupernatant samples taken from 71 to 115 hours in strain M236 (no addedprotease inhibitors). Only the CBHI carrier protein could be detectedaccumulating in the supernatant during that time. The same strain grownin the presence of chymostatin and pepstain inhibitors produced up to 9g/L of CBHI-BVS857 fusion at 104 hours. The BVS857-CBHI fusion proteinwas detected at 75 kD. The expression level determined was 3.0 g/L at 71hours, 6.3 g/L at 92 hours, 4.9 g/L at 98 hours, 9.0 g/L at 104 hours,and 7.8 g/L at 115 hours. The CBHI carrier levels increased throughoutthe culture and was maximal at 115 hours. Thus, the improvement inBVS857-CBHI production levels was dramatic and due to proteaseinhibition.

With the use of 40 g/L of cellulose production levels of 7.0 g/L wasreached at 101 hour for BVS857-CBHI and with 80 g/L of celluloseproduction levels were 9.0 g/L at 104 hours. This demonstrates thathigher production levels are achieved by adding more cellulose to theculture medium.

CBHI-streptII tag-3×(GGGS)-TEV site-BVS857 fusion protein from M236 waspurified via strep-tag affinity column (IBA GmbH) according themanufacture's protocol. The culture supernatant (600 μl) was applied tothe column and washed with 10 volumes of wash buffer. The column waseluted with elution buffer containing 2.5 mM desthiobiotin. Thefractions were run on a 4-20% criterion gel and stained with gel codeblue Coomassie strain. The eluted fractions #2 and #3 containedconcentrated protein that was slightly less than 75 kD, where the BVS857fusion protein should run.

The eluted fraction #2 was used for testing TEV protein cleavageefficiency. The AcTEV protease (Invitrogen, catalog#12575-015) was usedfor overnight incubation at 8° C. with different amounts of enzyme.Standard amounts of IGF1 were used on the immunoblot to quantify theamounts IGF1 present in each reaction. The amount of TEV was varied toevaluate its effectiveness (0, 1 μl, 5 μl, 10 μl, or 15 μl). TEV (5 μl)plus BVS857 control (4 μg) showed that there did not seem to be anysignificant TEV protease activity against BVS857. The baseline amount ofIGF1 in the carrier fusion sample was 687 ng. Using 1 μl (10 units) TEVenzyme converted 46% of the fusion after 16 hours at 8° C. The 5 μl (50units) amount converted about 95% of the fusion. This peaked at 97% when10 μl (100 units) of TEV was used. The TEV enzyme gave a backgroundstaining in the immunoblots at 25 kDa. The released BVS857 product atthe size of the native IGF1 could be easily observed around 10 kDa, butwas below the standard curve range. PMSF was used in the reactionbuffer, but it did not seem to neutralize the protease activityresponsible for degrading the BVS857 after it was released from thecarrier. The strain producing this material had only 2 proteasedeletions so protease inhibitors were used to try to control theprotease activity. This experiment showed that the internal strepII tagcan be used to affinity purify the CBHI fusion protein from supernatantand the TEV cleavage site is efficiently cut in order to release themodel protein such as IGF1.

Example 17 FGF21 Expression Strains in the 13-Fold Protease DeletionStrains

The first FGF21 production strain was made in M369(Δpep1Δtsp1Δslp1Δgap1Δgap2, see Example 4 of WO2013/102674) using vectorwith an FGF21 expression cassette targeted to the cbh1 locus, whichcontained a hygromycin marker. The vector was digested with PmeI andprocessed for transformation into the M369 strain, and transformantswere selected for with hygromycin. The expression construct produced aCBHI carrier-NVISKR kex2 site-FGF21 fusion protein, so that the secretedprotein would be the free FGF21 protein. Correct integration into thelocus and absence of the cbh1 orf was checked by PCR.

The CBHI carrier sequence was the same as for IGF1 above, followed byNVISKR Kex2 cleavage site and FGF21 sequence: SEQ ID NO. 926.

The M393 strain was grown with and without protease inhibitors pepstatinA, chymostatin, or soybean trypsin inhibitor. Independent wells werechosen for control wells where no inhibitors were added. This strain wasgrown in 3 ml of TrMM with diammonium citrate without ammonium sulfate,100 mM PIPPS, 20 g/L spent grain extract, 40 g/L lactose adjusted to pH4.5. The 24 well plates were shaken at 800 rpm at 85% humidity. Theplate was covered with an air permeable membrane and the cultures weregrown for 6 days.

Inhibitors were added first on day 3 and then added daily until day 6.200 μl samples were taken from the culture wells beginning on day 3. Themycelium was spun down for 5 minutes at 13 k and the supernatantcollected. From the culture supernatant 5 μl plus LSB was loaded in a4-20% SDS PAGE gel and immunoblotting made on nitrocellulose with rabbitanti-FGF21 (2 μg/ml) and goat anti-rabbit IgG AP conjugated secondaryantibody diluted 1:10,000. FGF21 standard were included on the blot forquantification.

FGF21 was very sensitive to protease degradation. On day 6 withouttreatment only about 2 mg/L of total protein could be produced of whichvery little was full length material (FIG. 12). With 10 μM pepstatin Aadded to the cultures the production could be improved up to 160 mg/L onday 6. In the presence of inhibitor a major degradation product around18 kD was detected. Adding SBTI or chymostatin, in addition to pepstatinA, did not produce an additional benefit. SBTI addition resulted in amuch more acidic culture sample and thus produced a negative effect onthe production level. Combination of pepstatin A and SBTI provided themost benefit as it allowed for 204 mg/L of total FGF21 to be produced.The expression levels were in the 1-12 mg/L range in the presence ofsingle inhibitors and untreated cultures reached about 1 mg/L. It wasimportant to inhibit both the aspartic proteases and the serineproteases to effectively produce FGF21.

The M393 strain was cultured in the fermentor in TrMM plus 2% yeastextract, 4% cellulose, 8% cellobiose, 4% sorbose, pH 4.5 withtemperature shifted from 28° to 22° at 48h. When FGF21 expression wasanalysed via immunoblotting the highest expression level was seen on day3 where there was a 10 kD degradation product produced at 130 mg/L. Nofull length FGF21 was observed. The FGF21 was very protease sensitivewhen produced in the 5-fold protease deletion strain. More proteaseactivity reduction would be required via inhibitor treatment or proteasedeletions. When inhibitors were used in 24 well cultures lots of proteinwas observed, suggesting FGF21 seemed to be well secreted but was justsensitive to degradation.

An FGF21 expression vector with AmdS marker was transformed into thestrain M1076 and M1085 (slp7 deletion with pyr4-hygromycin doublemarker).

The pTTv470 vector was created by taking the AmdS marker as a NotIfragment from pTTv249 and adding it to the previously made pTTv174 FGF21expression vector. The selection marker was simply exchanged to createthe new vector. The expression cassettes in pTTv174 and pTTv470 wereidentical. The pTTv470 vector was digested with PmeI, the expressioncassette was gel purified, and the FGF21 expression cassette with AmdSmarker was transformed into the two of the 13-fold protease deletionstrains M1076 (pep9 deletion) and M1085 (M901 with slp7 deletion).Following the AmdS selection, the resulting transformants were screenedby PCR for correct 5′ and 3′ integration and presence or absence of cbh1open reading frame. The primer pairs used are shown below in table 17.1.

TABLE 17.1 Primers for screening integration into the cbh1 orf. 5′integration, T095 + T096, ~2.8 kb (2776 bp), 58° C.T095_Ann112_F_cbhI_Ben GCTGTTCCTACAGCTCTTTC  (SEQ ID NO: 927)T096_Ann113_R_cbhI_Exon_Ben AGCCGCACGGCAGC (SEQ ID NO: 928) 3′integration, T008 + T022, ~1.9 kb, 60° C. T008_pHHO1-CBHIloc_GGTTGACTTACTCCAGATCG  cbh13′flankOutRev (SEQ ID NO: 929)T022_Amds_start_rc_seg CTGAAGCAACAGGTGCCAAG  (SEQ ID NO: 930)ORF, T1720 + T1721, gives a PCR band of 770 by if the cbh1 gene isnot deleted. Pure transformants do not give signal. 68° C.T1720_cbh1intronfor CCTGACGCTATCTTCTTGTTGG (SEQ ID NO: 931)T1721_cbh1intronrev CGCGCATGTTTGTCCATCAAAC (SEQ ID NO: 932)

Positive transformants were found for both transformed strains. TheM1076 transformation produced 13 good transformants and the M1085produced 2 good transformants. These transformants were cultivated in 24well cultures and compared to the earlier FGF21 production strain M393.The transformants and the control were grown in 10 g/L yeast extract, 20g/L cellobiose, 10 g/L sorbose with PIPPS, pH 4.5. The cultures werestarted by adding 1×10⁷ spores into 50 ml of culture medium and adding 3ml of culture medium plus spores to each well (6×10⁵ spores/well). 100μl of each culture supernatant was collected on days 4, 5, and 6. Themycelium was spun down and orange LSB was added to the supernatant andthe samples were heated for 5 minutes. 5 μl and 2 μl of thesupernatant+LSB was loaded per well into 4-20% Criterion TGX gel. Thegels were run and the proteins were transferred to nitrocellulosemembrane.

The expressed FGF21 was detected by immunoblotting. The rabbitanti-FGF21 antibody was diluted to 2 μg/ml in TBST. The stockconcentration provided was 3600 μg/ml (from Novartis). The goatanti-rabbit secondary IRDye 680 was diluted 1:30,000 in TBST. Theprimary and secondary antibody incubations were done for 1 hour at roomtemperature with shaking To detect the carrier CBHI levels the anti-CBHIantibody mab261 was diluted 1:10,000 in TBST. The goat anti-mouse IRDye800 secondary antibody was diluted 1:30,000 in TBST. Washed themembranes with TBST for 1 hour and rinsed with TBS. The blots werescanned at 700 and 800 nm.

The day 4, day 5, and day 6 samples were analysed for FGF21 and CBHIexpression. The difference between the M393 control strain and the newtransformants with 13 protease deletions was dramatic. The secretedFGF21 was primarily observed as a 10 kD product. This product was onlyfaintly visible in the M393 supernatants from day 4, but was massivelyexpressed in supernatant from all of the 13 new transformants (FIG. 13).On later days there was no FGF21 detected from the M393 strain. Therewas free CBHI carrier expressed in all the M393 control samples as wellas in the new FGF21 transformants. This indicated that all the strainssecreted both the CBHI carrier as well as the FGF21 protein, but therewas too much protease activity for the FGF21 in the M393 strain. Theeight additional proteases deleted in the new transformants dramaticallyimproved the stability of the FGF21. Four of the transformants #24, 36,48, and 73 seemed to produce higher amounts of CBHI and FGF21 comparedto the nine other transformatants. The difference was particularlyobvious on days 5 and 6. After Southern analysis this was determined tobe due to multiple integration of the FGF21 expression cassette. Thelower expression levels correspond to single copy integration. The M393strain has 5 protease deletions, while the new strains produced have 13proteases deleted. The transformants from the transformation of theM1085 strain were done in parallel. The results were similar as thoseseen in FIG. 16. The M1085 based strain also produced FGF21 productmainly at 10 kD, but there was a weaker 17 kD product visible on day 4.

Southern blot analysis was done on the several transformants.Transformant numbers #20, 24, 36, 48, 55, 73 from the M1076 strain and#4 and 39 from the M1085 were analysed via Southern blot usingradioactive detection. The genomic DNA was digested with PstI forintegration checking. Two PCR fragments were used as the probes for thecbh1 promoter and for the cbh1 3′ flank. The primers used to create thefragments are listed below.

cbh1 promoter probe, 799 bp fragment, 35 ng/μl (tube labelled 5-probe)T173_pcbhl_seq_r1 CAAAGGCCGAAGGCCCGAGG (SEQ ID NO :933) T1679CAACCTTTGGCGTTTCCCTG (SEQ ID NO :934) cbhl 3′flank probe, 796 bp fragment, 32.2 ng/μl (tube labelled 3′ probe)T178_cbh13flank_seq_f2 GGCCGCAGGCCCATAACCAG (SEQ ID NO: 935) T1680TGAGTGGGGGATGACAGACA (SEQ ID NO: 936)

The PstI digest and detection with both probes should give two bands at3.9 kb and 4 kb if correctly integrated into the cbh1 locus. Effectivelythere will only be one band at 4 kb. The native cbh1 locus gives asignal around 6.7 kb. The transformant numbers 20, 55, 4, and 39 showedthe expected integration band at 4 kb. These look to be clean singleintegrations into the cbh1 locus. As seen from the 24 well culturestudies, these transformants displayed a lower expression level thanmany of the other transformants. The highest expression levels were seenfrom transformants #24, 36, 48, and 73, which coincidentally showedextra integration signals in the Southern blotting. There was anonspecific band in all the transformants at 2.3 kb.

The multiple copy containing transformants #24 and #48 were named M1200and M1201. The single copy strains from transformant #20 and #55 werenamed M1202 and M1203. From the M1085 based strain transformants #4 and#39 were named M1204 and M1205, which were single copy strains.

The FGF21 production strains were cultivated in the 1 L fermentors withand without protease inhibitors. Cultivations were done with M1200,M1201, M1204, and M1205 strains. These strains were cultivated in TrMMwith 20 g/L yeast extract, 40 g/L cellulose, 100 g/L cellobiose, 40 g/Lsorbose at pH 4.5. Pepstatin, chymostatin, and SBTI inhibitors wereadded to the M1200 and M1205 cultures on day 3, 4, and 5. Thesupernatant samples were diluted in orange sample buffer so that 0.1 μlof supernatant could be loaded per well into a 4-20% Criterion PAGE gel.

The expressed FGF21 was detected by immunoblotting as above.

The cultivation of M1200 primarily produced a 10 kD product, but therewere higher molecular weight products up to 17 kD (FIG. 14). The fulllength FGF21 ran at 20 kD. On day 5 there was 2.0 g/L of the 10 kDproduct. Use of inhibitors increased the stability of the secretedFGF21. The major product was now 17 kD and produced up to 2.0 g/L on day5. With inhibitor treatment the full length version of FGF21 could beproduced at 0.2 g/L. This appeared to be full length. Cultivation ofM1205 produced a 10 kD band at 2.4 g/L on day 5 Inhibitor treatmentincreased the stability of the FGF21 product to generate a predominate17 kD form at 3.5 g/L and a full length product at 0.2 g/L on day 4(FIG. 14). Samples from the cultivation of M1201 had a technical problemwith the fermentor system (analysis not shown). The M1204 strain wasanalysed and gave similar expression levels, 2.5 g/L, of the 10 kDproduct on day 5. The 10 kD product was lowest on day 2 and was higheston day 5.

The inhibitor treatment demonstrated that it would be possible to fullystabilize the FGF21 protein. Further protease deletions to produce FGF21(without added protease inhibitors) include but are not limited to slp2,pep8, and pep11.

Inhibitor Studies on FGF21 Production Strain

To investigate which classes of proteases are contributing most to thedegradation of the FGF21 protein individual protease inhibitors wetested in 24 well cultures.

Chymostatin and SBTI are known to inhibit SLP2 and SLP7 subtilisinproteases that are still expressed in the M1200 strain. Pepstatininhibits aspartic proteases such as PEP8, PEP11, PEP12 which are knownto be secreted into the supernatant. 1,10-phenathroline targets mainlyzinc metalloproteases which are secreted by Trichoderma. The M1200strain was cultivated in 24 well format in TrMM with 10 g/L yeastextract, 20 g/L cellobiose, 10 g/L sorbose, PIPPS buffered at pH 4.5.Chymostatin, phenathroline, and pepstatin were used at a finalconcentration of 10 μM and SBTI was used at 0.1 mg/ml. Each treatmentwas done in duplicate wells and the untreated control was cultured in 4wells.

The cultures were started by adding 1×10⁷ spores into 50 ml of culturemedium and adding 3 ml of culture medium plus spores to each well (6×10⁵spores/well). 100 μl of each culture supernatant was collected on days4, 5, and 6. The mycelium was spun down to remove only the supernatant.Orange LSB was added and the samples were heated for 5 minutes. 2 μl ofthe supernatant+LSB was loaded per well into 4-20% Criterion TGX gel.The gel was run and proteins were transferred to nitrocellulosemembranes.

The expressed FGF21 was detected by immunoblotting as above. Acommercially purchased N-terminal antibody rabbit polyclonal from Abcam(#ab66564) and C-terminal rabbit polyclonal antibody from Abcam(#ab137715) were used in some cases.

Without inhibitor treatment the M1200 strain produced mainly a 10 kDproduct, but there were some larger forms visible in the day 4 samples(FIG. 15). Chymostatin treatment improved the stability of the proteinallowing a 17 kD major product. There were small amounts of the fulllength form visible and a dimer form around 37 kD after chymostatintreatment. Pepstatin, SBTI, and 1,10-phenathroline treatment onlyimproved the expression level of the 10 kD form. However, whenchymostatin and pepstatin were combined there was a synergetic effect onthe stabilization. The combined treatment promoted almost completestabilization of the full length form and reduced the lower molecularweight products. This was most apparent in the 6 day culture samples,where mainly the full length FGF21 could be visualized. The appearanceof the dimer was strongest with this treatment. Proper formation of thedimer indicates that the molecule has a C terminal cysteine residueneeded for dimerization.

The culture samples were probed also with an N- and C-terminal antibody.The dual treatment with chymostatin and pepstatin generated a fulllength product that reacted to the N-terminal antibody as well as theFGF21 standard (FIG. 15). When the C-terminal antibody was used todetect FGF21 produced in the 5 day samples two products were observedwhen treated with chymostatin or chymostatin/pepstatin (FIG. 16). Theseappear to be the full length protein and the lowest 10 kD form.Chymostatin treatment was enough to stabilize the C-terminus of theprotein. To maintain the N-terminus both chymostatin and pepstatin werenecessary.

The data from the day 4 immunoblots was quantified to make comparisonsof which inhibitors were most effective. The 1,10-phenathrolinetreatment best improved the total amount of FGF21 produced, thusindicating that zinc metalloproteases were likely involved (Table 17.2).There are many candidates such as mp1, mp2, mp3, mp4, and mp5. The fulllength form seems to be degraded by a subtilisin and an asparticprotease. The SLP2 protease is most likely to be the primary problem forthe stability and can be addressed by, for example, deleting the gene,silencing the gene, or switching the promoter of the gene. Deleting orsilencing aspartic proteases PEP8, PEP11, and PEP12 found in thesupernatant may further increase the stability of FGF21. In someembodiment, full-length production of FGF212 may only need deletion oftwo proteases, slp2 and pep8, in the strain M1200.

TABLE 17.2 Fluorescent units of FGF21 expression measured from the day 4immunoblot shown in FIG. 15. lower band upper band total productuntreated 27350 1521 28871 chymostatin 19500 34200 53700 pepstatin 510501610 52660 chymostatin/pepstatin 13100 49300 62400 SBTI 37050 1380050850 SBTI/pepstatin 35850 20400 56250 1,10-phenathroline 68800 77469574

Example 18 Reducing slp2 Expression by Replacing its Promoter

slp2 affects in some level growth and sporulation and therefore slp2promoter was replaced with a promoter which is expressed in lower levels(of all identified proteases slp2 mRNA was expressed the highest levelsin the conditions described in the last Example).

The promoters from the sporulation induced flavin containingmonooxygenase gene tre76230, the RNA polymerase gene tre49048, and theslp8 protease gene tre58698 were chosen for replacement into the slp2promoter locus. All genes appeared to be moderately expressed at levelsless than those seen with slp2.

A promoter region from the 3 genes was amplified by PCR and insertedinto a vector with slp2 flank sequences that would direct the promoterinto the correct position as the new slp2 promoter. The promotercassette contains the hygromycin marker upstream of the new promoter.The vectors were digested with PmeI, the replacement promoter constructswere gel purified, and transformed in the M507 MAB01 production strainunder hygromycin selection conditions. Two transformants from eachTransformants were isolated and named M773/M774, M775/M776, andM777/M778, respectively.

Promoter sequence for tre76230 promoter replacement: SEQ ID NO: 937.

Promoter sequence for tre49048 promoter replacement: SEQ ID NO: 938.

Promoter sequence for tre58698 promoter replacement: SEQ ID NO: 939.

These transformants and the M507 control strain were cultivated in 24well plates using TrMM with 1% yeast extract, 2% cellobiose, 1% sorbose,pH 5.5. Samples were taken on days 5, 6, and 7. Immunoblotting was donewith AP conjugate antibodies to detect the heavy and light chain. Theculture supernatants were diluted so that 0.5 μl was loaded per lane.Detection was visualized with AP substrate.

The heavy chain was clearly more stable in the M773/M774 and M777/M778transformants (FIG. 17). The most striking point is on day 7. The M507heavy chain is almost fully degraded, while the M773/M774 heavy chainsurvived very well. The other remarkable feature was that there was noupper degradation product at 37 kD. There are only some lowerdegradation products around 28 kD. The M773/M774 strains grew well, butthe M777/M778 strains were not growing that well and had troublesporulating. Thus, the preferred strain would be M773 or M774. Totalprotease activity measurements with casein correlated and indicated thatM773/M774 and M777/M778 have low protease activity.

When the light chains were detected via immunblotting there was lesslight chain in M773 and M774 compared to control. There was particularlyless light chain with M777 and M778 strains. The M775 and M776 lightchain amounts were similar to control. There seemed to be good agreementbetween the light chain amounts seen on the blots and the totalimmunoglobulin detected. The total IgG was measured from these culturesand showed that all of the promoter exchange strains had lower antibodyexpression, except for M775 and M776, which were very similar to control(Table 18.1). The M773/M774 strains had levels less than half M507.Thus, there might be something affecting the growth of some of thepromoter replacement strains, in this small culture format.

TABLE 18.1 Total antibody concentrations from 24 well culture day 7.Total mAB (protein G bound) Strain μg/ml M773 166 M774 225 M775 489 M776487 M777 68 M778 50 M507 control 503

The M774 and M775 strains were fermented. The two strains werecultivated in TrMM plus 20 g/l yeast extract, 40 g/l cellulose, 80 g/lcellobiose, and 40 g/l sorbose at pH 5.5 with the temperature shiftingfrom 28° to 22° at 48 hours for 10 days. The control strain M667 wascultivated under the same conditions and under similar conditions where120 g/l cellulose was used. Samples were taken for immunoblotting andtotal immunoglobulin determination. The supernatants were diluted sothat 0.05 μl was loaded per well in a 4-15% gel Immunoblotting to detectthe light chain was done with an AP conjugated antibody (A3818) diluted1:10,000 in TBST. The heavy chain was detected with an anti-human Fcantibody IRDye 700 DX conjugate (Rockland #609-130-003) diluted 1:30,000in TBST. The fluorescence at 700 nm was detected using an Odyssey CLxnear infrared imager (Li-Cor).

The MAB01 heavy chain produced from M774 looked remarkably good (FIG.18). The results were similar to those seen in the 24 well cultures.There was only one major degradation product around 25 kD. Typically,the heavy chain looks like that produced by M775. Normally there aredegradation bands at 38 kD, 28 kD, and 10 kD. The disappearance of the10 and 38 kD products may be explained by reduction of SLP2 activitybecause there are subtilisin cleavage site closer to the N terminus ofthe MAB01 heavy chain and without high slp2 expression the heavy chainis no longer cleaved at that position. The 28 kD product comes whenproteases cleave the heavy chain in the hinge region. The cleavage sitesin the hinge are more typical of sedolisin or trypsin like proteases.They don't appear to be caused by SLP2. The 10 kD product results whenthe hinge and the near N terminal sites are both cleaved.

No obvious differences were seen between the M774 and M775 light chainamounts or carrier bound percentages. While M775 is not optimal controlstrain, it resembles M507 very much, as seen in the 24 well culture datadescribed above. This data also suggests that SLP2 does not appear to becleaving the carrier-light chain fusion. Otherwise the amount of carrierbound material would increase dramatically when the slp2 expressionlevel was reduced.

M774 strain slightly outperformed M775 when the total antibody amountsare compared (Table 18.2). The total antibody amounts are also listed onthe heavy chain immunoblots. The biggest difference can be seen on day 7and 8, where M774 produced 5.3 g/L on both days. The M775 produced 4.4and 4.6 g/L on those days. The most important difference was observedonce the amounts of full length antibody are measured and compared. TheM774 produced a far more intact heavy chain. On day 7, the M774 strainproduced 71% full length, while the M775 was 37% full length. The M774strain could maintain 65% full length antibody up to 10 days, whereasthe M775 strain produced only 14% full length material. In terms ofquality, the control strain M667 (with a slp2 gene silencing construct)was similar to M774 (Table 18.2). However, the M667 production strainproduced slightly higher amounts of total antibody, at levels reaching6.1 g/L on day 10 under the same culture conditions. With increasedcellulose (120 g/L) the M667 strain could produce as high as 7.1 g/L oftotal antibody on day 10 (Table 18.2).

From these data, another approach to reduce SLP2 activity was generated.Replacing SLP2 promoter led to less expression of the SLP2 protease andresulted in remarkably low heavy chain degradation. Some effects ongrowth and sporulation were still observed, but they were far milderthan those seen with the complete deletion of the SLP2 gene. By givingslp2 a sporulation induced promoter it may have addressed thissporulation related problem. Any growth defect seen in M774 can becompensated with modifications in fermentation processes.

TABLE 18.2 Total antibody and full length antibody quantitation fromfermentor cultivation supernatants of strains M774, M775, and M667cultivations. Total mAB (protein G Full-size mAB Sample bound) (gelfiltration) Fermentation (day) μg/ml μg/ml % M774 5 3704 — — 6 4542 — —7 5301 3776 71 8 5290 3583 68 9 5453 3548 65 10 5455 3550 65 M775 5 3832— — 6 4263 — — 7 4436 1631 37 8 4641 1222 26 9 5152  974 19 10 5060  69014 M667 6 4895 — — 8 6005 — — 10 6070 — — 11 6248 — — 12 6676 — — 136435 — — 14 6175 — — M667 6 5828 — — 7 6181 4024 65 8 6674 4331 65 96885 4265 62 10 7063 4171 59 11 6690 — — 12 4674 — —

The M507 parental strain was compared to the M646 slp2 deletion strainand the M774 slp2 promoter switch strain in a separate fermentorcultivation series. They were grown in TrMM plus 20 g/l yeast extractand 120 g/l cellulose with 50% glucose/12.5% sorbose feed at pH 5.5 withthe temperature shifting from 28° to 22° at 48 hours Immunoblotting withanti-heavy and anti-light chain antibodies was done from the day 5 andday 6 samples to visualize the quality and relative amount of heavy andlight chain produced.

The M507 started growing a faster than the M646 and M774 strains, butthey caught up to M507 later in the culture. There was no significantdifference in the amount of free light chain produced by these threestrains, as seen on the immunoblot. There was a slightly higher amountof light chain bound to the CBHI carrier in the M646 strain. A smallamount of carrier bound heavy chain was detected from the M646 samplesand even less was seen in the M774 strain. This could not be detected inthe M507 strain. This observations suggests that SLP2 may be involved inprocessing the CBHI carrier-antibody fusion proteins to some extent inthe supernatant or potentially intracellularly.

The M774 strain showed a similar heavy chain degradation pattern to theslp2 deletion strain M646. Both strains were lacking the 10 and 38 kDdegradation products that were seen in the M507 parental strain. In theimmunoblot the M646 and M774 heavy chains showed only the full sizedproduct at 50 kD and one major degradation product at 25 kD. The amountof full length heavy chain in the M646 and M774 strains was highercompared to M507. The control M507 heavy chain showed two additionaldegradation products at 10 and 38 kD. This shows that indeed SLP2protease was responsible for producing these products. The amount oftotal MAB01 antibody produced on the day 6 time point was similarbetween the M774 strain and M507 strain, reaching 2.8 g/L under theseculture conditions (Table 18.3). Even though the M774 and M507 strainsproduced the same amount of total immunoglobulin, the quality of theM774 material was much better considering the lack of heavy chaindegradation products.

TABLE 18.3 Fermentation data from cultivation of M507, M646, M774. Thetotal antibody titers are shown in the table as g/L. Day M507 (g/L) M646(g/L) M774 (g/L) 3 1.6 1 0.8 4 2.3 1.4 1.5 5 2.7 1.8 2.2 6 2.8 2 2.8

Example 19 14-Fold Protease Deletion Strain—slp2 (tre123244) PromoterSwitch to tre76230 Promoter

To generate a 14-fold protease deletion strain with reduced SLP2activity, the 13-fold protease deletion strain M1077 (pyr4− of M1076)was transformed with MssI fragment of a plasmid targeted to the slp2(tre123244) locus to replace slp2 promoter with sporulation induced genetre76230 promoter.

Transformation was carried out using standard protoplast transformationmethod for pyr4 selection. Transformation was scaled to 2× (i.e. using500 μl protoplasts). Total amount of DNA used was ˜14.6 μg. Coloniesgrowing on transformation plates were picked on selective plates andscreened for correct integration of the deletion cassette using primersshown in Table 19.1. Selected clones giving integration signals werepurified via single spore purification and rescreened. Clones from threetransformants seemed to be pure after one purification round with strongintegration signals. Two pure sibling clones from each transformant wereplated onto PD+1 M sorbitol for spore suspensions.

TABLE 19.1Primers used in screening correct integration of the deletion cassette to the genome and change of slp2 promoter(tre123244) with tre76230 promoter. Primer SequenceT1729_slp2_int_check_F GACACTCCCTTGACTGTAGG (SEQ ID NO: 940)T488_pyr4_5utr_rev GGAGTTGCTTTAATGTCGGG (SEQ ID NO: 428) T1584_76230_f2CATCCCCCAAAGATGATGC (SEQ ID NO: 941) T1585_slp2_3int_orf_rTGTCATCGAGAGCAGAAGCA (SEQ ID NO: 942) T1393-slp2-5utr-FCACAACGTACTCGAAGTACC (SEQ ID NO: 943) T1586_slp2_promch_rAGGTCACTCAGCCTTGTACC (SEQ ID NO: 944)Southern Analyses of slp2 Promoter Replacement Strains

Three biological clones with two replicates of the slp2 promoterreplacement transformation along with their parental strain M1076 werecultivated for DNA extraction in 24-well plate. After three days myceliawere harvested by vacuum filtration, frozen and lyophilised. Genomic DNAwas extracted from mycelia using Easy-DNA kit (Invitrogen) and sampleswere analysed by PCR using the primers in Table 19.1. None of the clonesgave strong signal for slp2 promoter, but a very low amount of productcorresponding to expected size was seen for a few clones.

Genomic DNA from M124, M1076 and six Δslp2 promoter replacement strainswere digested with HindIII for all analyses (Δslp2 promoter, 5′ and3′flank). Strain M124 was omitted from flank analyses. A control plasmidwas digested with MssI for both flank analyses. Primers used to generateprobes for hybridisations are shown in Table 19.2.

TABLE 19.2 Primers used to produce probes for Southern analyses of slp2 (tre123244) promoter change with tre76230 clones. Primer Sequence SizeTarget T1393-slp2-5utr-F CACAACGTACTCGAAGTACC 541 bp slp2 promoter(SEQ ID NO: 945) T1586_slp2_promch_r AGGTCACTCAGCCTTGTACC(SEQ ID NO: 946) T1764_slp2_5fprobe_for TCAGATGGAGTCCCTTGAAC 647 bpslp2 5′ flank (SEQ ID NO: 947) T1765_slp2_5fprobe_rev CTGAATCTTGCTGGTCCG(SEQ ID NO: 948) T1766_slp2_3fprobe_for CAGCACATTCCAGATTGGC  853 bpslp2 3′ flank (SEQ ID NO: 949) T1767_slp2_3fprobe_revTGCTCAATGTGGGAGAGAGC (SEQ ID NO: 950)

The Southern analyses confirmed that all clones were pure slp2 promoterchange clones. In addition, majority of the clones give only theexpected signal with 5′ and 3′ flank probes verifying single integrationof the deletion cassette to the genome. One clone (78-28B) may have anextra copy of the cassette integrated to the genome. Clone 78-6A hasbeen stored for collection and designated with the code M1162. Cloneswith slp2 promoter change appear to have somewhat delayed and lowerability to sporulate.

Fermentor Cultivation of M1162

The M1162 strain was cultivated and compared to the strain M1076. Thepreculture was grown for 3 days, instead of the normal 2 days. The M1162was fermented in TrMM with 20 g/L yeast extract, 40 g/L cellulose, 80g/L cellobiose, 40 g/L sorbose at pH 4.5. The M1162 strain grew a bitslower than its predecessor M1076. The CO₂ peak for M1076 came at 75hours while the Ml 162 was at 94 hours.

Protease Activity Measurements

The protease activity from the fermentor cultivation samples wasanalysed. The total protein concentrations from the cultivation sampleswere measured so that the samples could be adjusted to 1 mg/ml totalprotein. 100 μl of all the diluted supernatants was added into 96 wellplate. Three replicate wells were used per sample. 100 μl of casein FLdiluted stock (10 μg/ml) made in sodium citrate buffer pH 4.5 was addedto each well of supernatant. The casein stock solution from the vial was1000 μg/ml and initially resuspended in 200 μL of PBS. For each sample abackground control was used with 100 μl of diluted supernatant and 100μl of sodium citrate buffer pH 4.5. The plates covered in plastic bagswere incubated at 37° C. The fluorescence from the plates was measuredafter 2, 3, and 4 hours. The readings were done on a fluorescent platereader using 485 nm excitation and 530 nm emission.

The protease activity of the supernatant coming from the M1162 strainwas extremely low, compared to the M1076 strain. The SLP2 proteaseactivity was seriously affected by switching the slp2 promoter. Theresulting protease activity upon casein was 3.6 times lower on day 3 and2.5 times lower on day 4 (Table 19.3).

TABLE 19.3 Protease activity measurements upon casein substrate forM1076 and M1162 fermentation supernatants. Samples were diluted so that1 mg/ml of total protein was used per sample. Casein FL substrate wasadded to measure the protease activity. Day M1076-13 deletions (units)M1162-14th round (units) 2 8.8 3.0 3 11.6 3.6 4 10.5 4.2 5 20.2 3.7

Example 20 Generation of 13-Fold Protease Deletion Strain ExpressingIFN-α 2b

The 13-fold deletion strain expressing interferon alpha 2b was createdin two steps. First, an interferon producing strain from M893 wasgenerated as described above, which contained 12 protease deletionsΔ(pep1 tsp1 slp1 gap1 gap2 pep4 pep3 pep5 pep2 sep1 slp8 amp2). The M893protoplasts were transformed with pTTv401 (derived from a plasmid havingcomplete cbh1 promoter and cbh1 terminator sequences but lacking CBH1encoding gene sequence inserted with IFN-α 2B, GGGGG-NVISKR) MssIfragment. The pTTv401 cassette carried the acetamide selection markerand was targeted to cbh1 locus.

M893 transformants were streaked on selection plates and PCR screeningfor correct integration into cbh1 locus. PCR screenings were done usingPhire Plant Direct kit (Thermo Scientific, F-130). Screening primers arelisted on Table 43.1. A strain was named M1012.

TABLE 20.1 Screening primers for pTTv401 into M893 transformation.Sequence 5′ -->3′ 5′ integration screening cbh1 locusT095_Ann1 12_F_cbhI_Ben GCTGTTCCTACAGCTCTTTC Product (SEQ ID NO: 951)~2.8 kb T096_Ann1 13_R_cbhI_Exon_Ben AGCCGCACGGCAGC (SEQ ID NO: 952) 3'integration screening cbh1 locus T008_pHHO1- GGTTGACTTACTCCAGATCGProduct CBHIloc_cbh13′flankOutRev (SEQ ID NO: 953) ~1.9 kbT022_Amds_start_rc_seg CTGAAGCAACAGGTGCCAAG (SEQ ID NO: 954)cbh1 ORF screening T685_Tdm_11_screen_ GCCTTTGGGTGTACATGTTTG 871 bp(SEQ ID NO: 1048) product if T908_CBH1_intron_rev TGGCCAGTCAGCTGGGAGCCCBH1 (SEQ ID NO: 955) orf still present

M1012 was confirmed by Southern analysis to carry one IFN-α 2bexpression cassette at cbh1 locus. M1012 spores were plated on 5-FOAplates to loopout pyr4 marker. Colonies were picked and streaked on5-FOA plates. Single cell plating was done for ten clones and subcloneswere screened by PCR to confirm pyr4 loopout. Screening primers arelisted in Table 20.2. Clones which gave correct signals for pyr4 loopoutwere streaked on PD plates and +/− uridine plate test was done forselected clones. M1012 pyr4 negative clone 1-1 was named as M1065 andpyr4 negative clone 2-1 was named as M1066.

TABLE 20.2 Primers for screening pyr4 loopout from M1012 Primer pair 1:T1556_amp2_5f_f2 AAGTGTGCTGATGTGATGGA (SEQ ID NO: 956) T1557_amp2_3f_r2GCATGCGAAGTACCTTACGA (SEQ ID NO: 957) Primer pair 2:T061_pyr4_orf_screen_2F TTAGGCGACCTCTTTTTCCA (SEQ ID NO: 382)T624_gpdA_seqR1 CTCCATATTCTCCGATGATGC (SEQ ID NO: 852)

To generate 13-fold protease deletion strain with IFN-α 2b expression,M1065 protoplasts were transformed with MssI fragment of slp2(tre123244) deletion vector pTTv457 (generated from pTTv115 byintroducing/changing pyr4-hygromycin selection marker). Protoplastingand transformation was carried out as above. pTTv457 M1065 transformantswere streaked on selection plates and PCR screened for correctintegration into slp2 locus. PCR Screening primers are listed on Table20.3. Three transformants which did not give slp2 ORF signal werestreaked on PD+1M sorbitol plates.

TABLE 20.3 Primers for screening slp2 deletion cassette integrationand slp2 ORF deletion. 5' integration screening T054_slp2_5screen_FGATGCACCGCTGCGGCC (SEQ ID NO: 327) T026_Pyr4_orf_5rev2CCATGAGCTTGAACAGGTAA (SEQ ID NO: 328) 3' integration screeningT047_trpC_term_end_F CCTATGAGTCGTTTACCCAGA (SEQ ID NO: 426)T055_slp2_3screen_R GGCGTTGCTCCCCATGCG (SEQ ID NO: 330) slp2 orfT1231_slp2 seqF2 AACGGATCCGGCACCATGTC (SEQ ID NO: 958) T112_slp2_ORF_RTTACTCGGAGAGCTCAGAGA (SEQ ID NO: 332)

The best clone was designated M1106 and cultivated with the controlstrain M961 in a 1 L fermentor in TrMM with 20 g/L yeast extract, 40 g/Lcellulose, 100 g/L cellobiose, and 40 g/L sorbose at pH 4.5. Theprevious standard amount of cellobiose was 80 g/L, so the M961 strainwas also cultivated in the control medium (TrMM with 20 g/L yeastextract, 40 g/L cellulose, 80 g/L cellobiose, and 40 g/L sorbose at pH4.5).

The culture samples were analysed via immunoblotting to quantitate theexpression of interferon. The samples were diluted so that 0.05 μl ofsupernatant could be loaded in 10 μl in a 4-20% criterion page gel.Standard amounts of interferon corresponding to 400, 200, 100, 50, and25 ng were also loaded to the same gel. The interferon antibody (Abcam#ab9386; diluted to 1 μg/ml in TBST) was incubated with the blotmembrane for 1 hour and washed with TBST. The secondary antibody IRDye680 (Li-Cor #926-68070; diluted 1:30,000 in TBST) was incubated for 1hour, washed with TBST, and scanned at 700 nm

In all the immunoblots interferon was detected as one band about 17 kD.There was a small amount of carrier bound interferon detected about 75kD, but the majority was in the free form. The M961 strain achieved aninterferon production level of 3.2 g/L at 95 hours (Table 20.2). In thepresence of higher concentration of cellobiose the interferon productionlevel reached 7.9 g/L at 95 hours and the expression was stable from 89hours through 99 hours where levels were 7.4 g/L or greater. To checkwhat the maximum production level would be if more proteases weredeleted, the M961 strain was grown in the same medium with chymostatin(20 μM) and pepstatin (10 μM) added on days 3-5. The protease inhibitortreatment raised the interferon production level to 10.7 g/L at 140hours. During M1106 cultivation the expression of interferon was seenfrom 90 hours up to 121 hours. The highest amount was measured to be 4.3g/L (Table 20.4). The strain M1106 grew somewhat slower: the M1106reached 4.3 g/L at 121 hours, whereas the M961 reached 7.4 g/L at 90hours.

TABLE 20.4 Expression level of interferon alpha 2b detected in thefermentor cultivations in g/L. time M961 (g/L) M961 (g/L) M1106 (g/L)70.75 h 2.3 3.4 89.75 h 3.1 7.4 90.4 h 1.1 95.1 h 3.2 7.9 96.4 h 1.7 99h 3.2 7.4 102.6 h 2.4 103.75 h 2.9 6.3 114 h 3.4 121.5 h 4.3 cellobiose:80 g/L 100 g/L 100 g/L

Example 21 Using CRISPR-CAS System to Generate Gene Deficient Strains ofT. reesei

Cas9 nuclease sequence with C-terminally tagged nuclear localizationsignal (nls) is codon optimized for expression in Trichoderma reesei.Sequence is cloned under the control of constitutive gpdA promoter andtrpC terminator sequences, using basic cloning vector and standardprocedures. Final Cas9 nuclease expression vector is constructed fromfollowing components: pep4 protease (or any other suitable protease)locus 5′ flanking sequence+pgpdA-Cas9-nls-ttrpC cassette+pyr4-hyg^(R)double selection cassette and pyr4 loop-out sequence+pep4 protease locus3′ flanking sequence. Vector is constructed to pRS426 backbone byutilizing yeast recombination methodology; overlaps between the vectorcomponents are generated with PCR primers. Cas9 nuclease expressionvector is transformed with peg-mediated protoplast transformation methodto wild-type T. reesei M124 strain or any other T. reesei straingenerated above or in WO/2013/174927 or WO/2013/102674, generatingsimultaneously pep4 protease deletion. Generated strain Cas9_M124 isthen used as a background strain for transfection of transient gRNAcassettes generated by PCR, as described in DiCarlo et al. 2013 (Genomeengineering in Saccharomyces cerevisiae using CRISPR-Cas systems; NAR41:4336-4343). Alternatively, RNA polymerase III SNR52 promoter- andSUP4 3′ flanking region from Saccharomyces are replaced with Trichodermahomologues. Guide RNA needed for precise genomic targeting of CAS9nuclease is located between the promoter and 3′ flanking region. GuideRNA is composed of 20 nt's long sequence complementary to desiredgenomic target, followed by 3 nt's complementary with NGG PAM(protospacer-adjacent motif)—sequence and constant 3′ portion requiredfor CAS9 activity. Exemplary guide RNAs are shown in Table 21.1 forvarious proteases and glycoenzymes harmful for heterologous proteinproduction. The genomic targets are selected among hydrolytic enzymes orenzymes from glycan biosynthesis pathway of Trichoderma reesei.Transient guide RNA cassettes (single and multiple) are introduced toCas9_M124 protoplasts by electroporation or by other basic gene transfermethod. Protease deficient clones are selected on the basis of reducedprotease activity, caused by CAS9-generated point mutations to desiredgenomic target sequences. Clones with point mutations targeted to glycanbiosynthesis pathway can be selected by glycan profiling. After singlespore purification, selected clones are characterized by PCRamplification of genomic target locus and sequencing of the PCR product,to verify the point mutation inactivating the gene.

Alternative way to produce guide RNA is to express the sequence ormultiple sequences from promoter transcribed by RNA polymerase II andflank the guide RNA's with self-processing ribozyme sequences, asdescribed in Gao and Zhao 2014 (Self-processing of ribozyme flankedRNA's into guide RNA's in vitro and in vivo for CRISPR mediated genomeediting; Journal of Integrative plant biology, 56:343-349). FIG. 19 showa phylogenetic tree of a subset of the proteases amenable to deletions.

TABLE 21.1 Guide RNA sequences targeted to T. reesei proteases and ALG3.Enzyme id Guide RNA sequence pep1  74156CCCCACCGAGGGTCAGAAGA (SEQ ID NO: 959) pep2  53961CACCGTCCTGTCTGCCTCCA (SEQ ID NO: 960) pep3 121133TCCAGGCCCAGGCAAAGTTC (SEQ ID NO: 961) pep4  77579GTTCAACGACAAGCCGCCCA (SEQ ID NO: 962) pep5  81004GCATGCCATTGAGATCAACC (SEQ ID NO: 963) pep7  58669CCACGCGCGGCGCCCCAAGC (SEQ ID NO: 964) pep8 122076ATTACGTTGCAGCTCGACAC (SEQ ID NO: 965) pep11 121306CACCACCTTTGTCGACGCCA (SEQ ID NO: 966) pep12 119876GACGCCATCAATAACCTCAC (SEQ ID NO: 967) pep9  79807CCCGATGCGCCCAACACCGC (SEQ ID NO: 968) tsp1  73897TCGCAGATCCGCGTCCGCGC (SEQ ID NO: 969) slp  57433ATCTATCTAAGCATTTCGCA (SEQ ID NO: 970) slp  35726GCTGCCCCTGATGCGACTAT (SEQ ID NO: 971) slp  60791GTCGACCAACTCCATACTCA (SEQ ID NO: 972) slp 109276AACGACACCGACATCTTCTA (SEQ ID NO: 973) slp1  51365CGCGTACATCTTCGAATTCG (SEQ ID NO: 974) slp2 123244CTGAAGCACACTTTCAAGAT (SEQ ID NO: 975) slp3 123234CTTGTTCCCACTACCAAGCA (SEQ ID NO: 976) slp5  64719ACTCCTTCAGCATGCACACC (SEQ ID NO: 977) slp6 121495AGAAACCGTTAAGCAGATCA (SEQ ID NO: 978) slp8  58698AACAAGAACAGCACGTTCGA (SEQ ID NO: 979) gap1  69555GTGATGGCACCTACGATGCC (SEQ ID NO: 980) gap2 106661GTGCTGCCCGCCGCTCCAAC (SEQ ID NO: 981) gap3  70927GTCATTGATTCGCCCCCAGA (SEQ ID NO: 982) gap4  57575CGCGAATTCCCCTCAGACTC (SEQ ID NO: 983) amp1  81070GAGCTTCTACAAGTTCGCAA (SEQ ID NO: 984) amp2 108592CCTCGACTCGCGCTTCGTCA (SEQ ID NO: 985) sep1 124051GCAGCCAGCACTCCCACCTA (SEQ ID NO: 986) slp7 123865TCTCCGACCCCTCAAGCCCA (SEQ ID NO: 987) tpp1/sed3  82623GCAGTTCTGCCGTCGAGTCT (SEQ ID NO: 988) sed2  70962GAGATACCAGCAACGCGCGA (SEQ ID NO: 989) sed5 111838GATCCTTCATCAGAAACATT (SEQ ID NO: 990) sed3  81517GCAGCCATATATCGACAGCC (SEQ ID NO: 991) mp1 122703CAGACGACGACGCTCAAGAA (SEQ ID NO: 992) mp2 122576GACGCTGCCTCATCTAGTCG (SEQ ID NO: 993) mp3   4308GCTGCGCGATCTCGACTTCA (SEQ ID NO: 994) mp4  53343CTCACATTCTCTATTCACGA (SEQ ID NO: 995) mp5  73809TGTGCTCCTGACCGACAAGC (SEQ ID NO: 996) ALG3 104121ACTGCCGTGGACATTGCCAA (SEQ ID NO: 997) a protease  80843CAGTCACCAGCAAGACAAAG (SEQ ID NO: 998) a protease  72612CGACCTCCACGATGTCATCA (SEQ ID NO: 999) a protease  47127AAGACGAAGCTCCGCCAATC (SEQ ID NO: 1000) a protease  77577AAGAGCACGACCGTTTCGTC (SEQ ID NO: 1001) pep13  76887TCTGACGCTGCTCCTCGCGA (SEQ ID NO: 1002) a protease  56920ATCACCGACACGCGAGACCT (SEQ ID NO: 1003) a protease 120998GTCGCTGGCCTCGTCCCTCA (SEQ ID NO: 1004) a protease  65735GAGCTCGTCCGACCCCCGCC (SEQ ID NO: 1005) a protease  82141CCCTGTCCCTGACCTTACAA (SEQ ID NO: 1006) a protease 121890GAAATCACAACACTGCCAAA (SEQ ID NO: 1007) a protease  22718GAACTCAACCTCCAAGACGC (SEQ ID NO: 1008) a protease  21659GAGTATGTTGCCATGTTCCT (SEQ ID NO: 1009) a protease  82452CACGGAGACTGCTGCCGCTC (SEQ ID NO: 1010) a protease  81115CTACTTCACCTACGACATCC (SEQ ID NO: 1011) a protease  64193CATCCTCACCATCCTCACCA (SEQ ID NO: 1012) a protease  23475GCTCTCACGAAATCCTCGAC (SEQ ID NO: 1013) a protease  79485GCTCTCTGAGCCTGCAAGAC (SEQ ID NO: 1014) a protease 122083CCGCGTCTCCTGCACGTAGT (SEQ ID NO: 1015) a protease  61127TGCGCGACCCCGTCATCGTC (SEQ ID NO: 1016) a protease  80762ACCTCTCTGGTCCACGACCT (SEQ ID NO: 1017) a protease  56853GACTCCTCCCTCCACACCGT (SEQ ID NO: 1018) a protease  22210TCTGTCGAGGAGAGCAACAT (SEQ ID NO: 1019) a protease 111694ACGCGCAACAACCGCCGCAC (SEQ ID NO: 1020) a protease  40199GCCCGACCGGTTCAACGTCC (SEQ ID NO: 1021) a protease  75159TTCGACAAGCTCACTTACAA (SEQ ID NO: 1022) a protease  21668CTTCGACTCCCACTCCAAGA (SEQ ID NO: 1023) a protease  61912CGCCGCTGCCCTCTTCGAAA (SEQ ID NO: 1024) a protease  58387CGTCACAGAGCACTTCTTCC (SEQ ID NO: 1025) a protease  82577CAGCACGACTCCATCTACGC (SEQ ID NO: 1026) a protease  81087AACCACATCGCCGAGAACAA (SEQ ID NO: 1027) pep10  78639CCTTGTCTATGCGAATGACC (SEQ ID NO: 1028) pep16 110490AGCAGCAGCAGCACGAGCAG (SEQ ID NO: 1029) pepl4 108686TCCACGTTTGAGCTGCGTGT (SEQ ID NO: 1030) pep6  68662ATCCCCATCCACCAGAAGCG (SEQ ID NO: 1031) a protease  66608CGTCTTCGACCGAATACAAG (SEQ ID NO: 1032)

Example 22 Transcriptome Analysis with Trichoderma reesei Strains M629and M507

Trichoderma reesei strains M629 (MAB01, pcDNA1-(Kre2)huGnt1,pgpdA-(nat)huGnt2, Δpep1 tsp1 slp1 gap1 gap2 pep4 pep3) (as described inExample 19 of WO2013/102674) and M507 (MAB01, Δpep1 tsp1 slp1 gap1 gap2pep4 pep3) (as described in Example 6 of WO2013/102674) were cultivatedin fermentor with standard Yeast extract—and Spent grain extract culturemedias. Total RNA was purified with standard methods, from samplescollected on days 1, 3 and 4 (Yeast extract media) and on days 1, 3 and5 (Spent grain extract media). mRNA was purified from total RNA sampleswith Machery-Nagel nucleotrap mRNA kit according to Kits instructions.Conversion to cDNA and preparing for sequencing was made with IlluminaTruSeq Stranded mRNA Sample Prep Kit. 250-450 base pare products werecollected for sequencing, with Illumina hiScanSQ sequencer(100+8(index)+100 cycles, paired end run).

For statistical analysis the sequence reads were manipulated asfollowing. The 9134 gene reads originating from the sequencing werecleaned (i.e. genes with all values with zero or average all overconditions below 0.1 were removed) and this resulted 8525 gene reads. Ofthe 8525 genes potential protease genes were identified based onsequence similarity to other identified Trichoderma proteases or theones of other filamentous fungi (Aspergillus, Neurospora). The followingproteases either show constant or regulated expression levels indifferent time points and/or culture conditions (based on FPKM values;fragments per kilobase of exon per million fragments mapped) and shouldtherefore be deleted: a metalloprotease mp1 (TR122703), a protease(TR80843), a peptidase (TR72612), a protease (TR47127), a peptidase(TR77577), pep13 (TR76887), a protease (TR56920), a carboxypeptidase(TR120998), a protease (TR65735), a peptidase (TR82141), ametalloprotease (TR121890), a peptidase (TR22718), a peptidase(TR21659), mp5 (TR73809), a protease (TR82452), a peptidase (TR81115), apeptidase (TR64193), a protease (TR23475), a peptidase (TR79485), mp3(TR4308), a protease (TR122083), a carboxy peptidase (TR61127), apeptidase (TR80762), a peptidase (TR56853), a peptidase (TR22210), aprotease (TR111694), mp4 (TR53343), mp2 (TR122576), a protease(TR40199), a protease (TR75159), amp2 (TR108592), a protease (TR21668),amp1 (TR81070), a protease (TR61912), a protease (TR58387), a protease(TR82577), a protease (TR81087), pep10 (TR78639), pep16 (TR110490), pep7(TR58669), pep14 (TR108686), pep6 (TR68662) and a protease (TR66608).

Example 23 Generation of mp1 or mp5 Deletion Strains

The deletion plasmid pTTv468 for the metalloprotease mp1 (tre122703) wasconstructed using yeast homologous recombination. The plasmid has mp1 3′direct repeat for looping out the pyr4 marker. A NotI digested plasmid(from a plasmid with hygr-pyr4 double marker derived from plasmidpTTv194) was used as backbone. mp1 3′ direct repeat and pyr4 marker wereconstructed by PCR using oligos listed in Table 23.1. pTTv468 vector wasassembled using yeast homologous recombination cloning as describedabove.

TABLE 23.1 Primers used for cloning deletion vector pTTv468for mp1 (tre122703). T1731 CAGCGTCGAAAGATTTCATCAAACCGCTCAATTGACCATCGCGGCCGCGATGCGAAGCGAATGGAG CA (SEQ ID NO: 1033) T1732GCGCTGGCAACGAGAGCAGAGCAGCAGTAGTCG ATGCTAGGGCGCGCCTATACCTCACAATAGACGGA (SEQ ID NO: 1034) T1369_pyr4_for CTAGCATCGACTACTGCTGC (SEQ ID NO: 1035) T1733 TTTTCAGACAAGTCCGCCCCTGCTCCATTCGCTTCGCATCGCGGCCGCGGCGCGCCATGCAAAGATAC ACATCAAT (SEQ ID NO: 1036)

To generate a 15-fold protease deletion strain, the 14-fold proteasedeletion strain M1199 (pyr4− of M1162) was transformed with MssIfragment of pTTv468. Transformation was carried out using standardprotoplast transformation method for pyr4 selection. Colonies growing ontransformation plates were being picked on selective plates and screenedfor correct integration of the deletion cassette using primers shown inTable 23.2.

TABLE 23.2 Primers used for screening. T1100_mp1_screen_5flk_fwdGTCTTGGCCATCAATGGAGT (SEQ ID NO: 1037) 488_pyr4_5utr_revGGAGTTGCTTTAATGTCGGG (SEQ ID NO: 428) T061_pyr4_orf_screen_2FTTAGGCGACCTCTTTTTCCA (SEQ ID NO: 382) T1101_mp1_screen_3flk_revACGGCTTACGAACAACGAGT (SEQ ID NO: 1038) T1102_mp1_orf_fwdACATCCTGGCCGATATTCTG (SEQ ID NO: 1039) T1103_mp1_orf_revGCTGTAGCTGGTGGAGAAGC (SEQ ID NO: 1040)

The deletion plasmid pTTv469 for the metalloprotease mp5 (tre73809) wasconstructed using yeast homologous recombination. The plasmid has mp5 3′direct repeat for looping out the pyr4 marker. mp5 3′ direct repeat andpyr4 marker were constructed by PCR using primers listed on Table 23.3.

TABLE 23.3 Primers used for cloning deletion vector pTTv469for mp5 (tre73809). T1668_mp5_ TTGCAAGTCGGACTCTGGACGCTTCGTGAA 3DR_fwATCCCCCGCAGCGGCCGCACATTAGAGCTC TCTCCTCC (SEQ ID NO: 1041) T1669_mp5_GCGCTGGCAACGAGAGCAGAGCAGCAGTAG 3DR_rev TCGATGCTAGGGCGCGCCTACGGCTGTCACAATGCACA (SEQ ID NO: 1042) T1369_pyr4_for CTAGCATCGACTACTGCTGC (SEQ ID NO: 1035) T1734 TACCCAGACGTAGAGAAGGAGGAGGAGAGAGCTCTAATGTGCGGCCGCGGCGCGCCATGC AAAGATACACATCAAT  (SEQ ID NO: 1043)

To generate another 15-fold protease deletion strain, the 14-foldprotease deletion strain M1199 (pyr4− of M1162) was transformed withMssI fragment of pTTv469. Transformation was carried out using standardprotoplast transformation method for pyr4 selection. Colonies growing ontransformation plates were being picked on selective plates and screenedfor correct integration of the deletion cassette using primers shown inTable 23.4.

TABLE 23.4 Primers used for cloning deletion vector pTTv469.T1677_mp5_5fl_int_Fw GAACCAGCGCTCCAATACCT  (SEQ ID NO: 1044)T488_pyr4_5utr_rev GGAGTTGCTTTAATGTCGGG  (SEQ ID NO: 428)T061_pyr4_orf_screen_2F TTAGGCGACCTCTTTTTCCA  (SEQ ID NO: 382)T1678_mp5_3fl_int_rev GGGCTGTGTGTGTGTGTTTG  (SEQ ID NO: 1045)T1675_mp5_orf_f GCCTGGTCGATACTGCTCTC  (SEQ ID NO: 1046) T1676_mp5_orf_rCCTGTTGGGTATGAAGGCGT  (SEQ ID NO: 1047)

Example 47 Generation of 13-Fold Protease Deletion Strain with slp3Deletion

To generate a 13-fold protease deletion strain with Δslp3, the 12-foldprotease deletion strain M901 (pyr4− of M893) was transformed with MssIfragment of pTTv425. Transformation and screening of the clones wereperformed as described above. One original transformant was obtained andafter three purification rounds clones were rechecked by PCR (see Table6-1). A subclone was named as M1075.

We claim:
 1. A Trichoderma reesei filamentous fungal cell comprising atleast one endogenous protease having reduced or no protease activity,and a recombinant polynucleotide encoding a heterologous polypeptide,wherein the gene encoding endogenous amp2 protease of SEQ ID NO:775comprises a mutation that reduces or eliminates the activity of theendogenous amp2 protease, and wherein the production level of theheterologous polypeptide is at least two-fold higher than the productionlevel of the same polypeptide as produced in a corresponding parentalfilamentous fungal cell in which the gene encoding endogenous amp2protease of SEQ ID NO:775 does not comprise a mutation that reduces oreliminates the activity of the endogenous amp2 protease.
 2. TheTrichoderma reesei filamentous fungal cell of claim 1, wherein the cellfurther has reduced or no detectable protease activity in at least threeor four proteases selected from the group consisting of pep1 of SEQ IDNO: 1, pep2 of SEQ ID NO: 182, pep3 of SEQ ID NO:17, pep4 of SEQ ID NO:37, pep5 of SEQ ID NO: 58, pep8of SEQ ID NO: 507, pep 11 of SEQ ID NO:522, pep12 of SEQ ID NO: 530, tsp1 of SEQ ID NO: 66, slp1 of SEQ ID NO:82, slp2 of SEQ ID NO: 98, slp3 of SEQ ID NO: 166, slp7of SEQ ID NO:231, gap1 of SEQ ID NO: 118, and gap2 of SEQ ID NO:
 129. 3. TheTrichoderma reesei filamentous fungal cell of claim 2, wherein the totalprotease activity is reduced to 40% or less, or 6% or less, of the totalprotease activity of the corresponding parental filamentous fungal cellin which the proteases do not have the reduced activity.
 4. TheTrichoderma reesei filamentous fungal cell of claim 1, wherein theheterologous polypeptide is a mammalian polypeptide selected from thegroup consisting of an antibody and its antigen-binding fragments, agrowth factor, an interferon, a cytokine, and an interleukin.
 5. TheTrichoderma reesei filamentous fungal cell of claim 1, wherein thefungal cell further comprises ALG3 having reduced activity.
 6. TheTrichoderma reesei filamentous fungal cell of claim 5, wherein the geneencoding ALG3 comprises a mutation that reduces or eliminates thecorresponding activity.
 7. The Trichoderma reesei filamentous fungalcell of claim 1, further comprising a) anN-acetylglucosaminyltransferase I catalytic domain, and, optionally, b)an N-acetylglucosaminyltransferase II catalytic domain.
 8. A method ofimproving the stability of a heterologous polypeptide, comprising a)providing the Trichoderma reesei filamentous fungal cell of claim 1, andb) culturing the cell such that the heterologous polypeptide isexpressed, wherein the heterologous polypeptide exhibits increasedstability compared to the heterologous polypeptide produced in acorresponding parental filamentous fungal cell in which the endogenousamp2 protease does not have reduced activity.
 9. A method of making aheterologous polypeptide, comprising a) providing the Trichoderma reeseifilamentous fungal cell of claim 1; b) culturing the cell such that theheterologous polypeptide is expressed; and, c) purifying theheterologous polypeptide.